Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

Embodiments of the invention provide a method of improving the efficacy
of an anti-cancer therapy and a method of treatment of cancer by
normalizing angiogenesis in cancer. By enhancing the cell signaling
pathway via a TRPV4 receptor in tumor endothelial cells, either by a
TRPV4 agonist or by increasing the expression of TRPV4 in the tumor
endothelial cells, the tumor endothelial cells behave normally and form
normal angiogenic network for better anti-cancer therapy to the tumors.

Claims:

1.-7. (canceled)

8. A method for increasing the efficacy of an anti-cancer treatment in a
patient in need thereof, the method comprising administering a TRPV4
agonist or a vector comprising a DNA sequence encoding TRVP4 to the
patient concurrently with a cancer treatment or subsequently
administering the cancer treatment to the patient.

9. A method for cancer treatment in a patient in need thereof, the method
comprising administering a TRPV4 agonist or a vector comprising a DNA
sequence encoding TRVP4 to the patient concurrently with a cancer
treatment or subsequently administering the cancer treatment to the
patient.

10. The method of claim 8, wherein the cancer treatment is chemotherapy,
radiation therapy or immunotherapy.

11. The method of claim 8, wherein the TRPV4 agonist is selected from a
group consisting of GSK1016790A, Bisandrographolide A (BAA), RN 1747,
AB1644034, α-phorbol 12,13-didecanoate (4.alpha.PDD) 5,6-EET,
acetylcholine and App441-1

14. The method of claim 9, wherein the cancer treatment is chemotherapy,
radiation therapy or immunotherapy.

15. The method of claim 9, wherein the TRPV4 agonist is selected from a
group consisting of GSK1016790A, Bisandrographolide A (BAA), RN 1747,
AB1644034, α-phorbol 12,13-didecanoate (4.alpha.PDD) 5,6-EET,
acetylcholine and App441-1

Description:

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit under 35 U.S.C. §119(e) of
U.S. Provisional Application No. 61/357,123 filed Jun. 22, 2010, the
contents of which are incorporated herein by reference in its entirety.

BACKGROUND OF INVENTION

[0003] It is well known that the new network of blood vessels occurs in
cancer and the network supplies nutrients that sustained the uncontrolled
growth of abnormal cells in the body. However, it has been shown that
these blood vessels are distinct from those of normal healthy tissues.
The differences can affect the delivery and therefore the efficacy of
anti-cancer therapy that targets the cancer cells, e.g, solid tumors.

[0004] The network of blood vessels and constituents in tumors has
abnormal structures and functions. For example, the network of blood
vessels have irregular morphology and pattern; the blood vessels tend to
be thicker and have large clumps of tumor endothelial cells (TECs), the
blood vessels are hyperpermeable ("leaky"), and the TECs have abnormally
high basal level of active Rho, increased rate of cell migration, and
aberrant mechanosensory response and orientation to external
mechano-stimuli such as stretch stress when compared to non-cancer
derived, normal endothelial cells (nECs). These abnormal TECs lead to
abnormal angiogenesis in tumors, resulting in the irregular networks and
"leaky" blood vessels. Many solid tumors show an increased interstitial
fluid pressure (IFP) due to the irregular network, which forms a physical
barrier to drug delivery, particular to the interior of a solid tumor.
For example, the hyperpermeability of the tumor blood vessels creates a
situation where a therapeutic effective amount of anti-cancer therapy
fails to reach the target area because a substantial amount of the
anti-cancer therapy has leaked out of the blood vessels enroute to the
interior of a solid tumor. In addition, the irregular network affects
blood flow rate and can impede a sustained delivery of an anti-cancer
therapy to the target area. Therefore, innovations that address the
abnormal angiogenesis, blood vessel network and abnormal characteristics
of TECs in cancer can potentially impact the effectiveness of anti-cancer
therapies.

SUMMARY OF THE INVENTION

[0005] Embodiments of the present invention are based on the discovery
that tumor endothelial cells (TECs) have abnormal characteristics and
responses compared to non-cancer or non-tumor associated normal
endothelial cells (nECs) Unlike nECs, TECs express abnormally low levels
of a stress-activated (SA) ion channel receptor TRPV4 compared to nECs
(FIGS. 1A and 1B). These TECs also have reduced calcium influx upon
stimulation of the TRPV4 receptor (FIGS. 1C and 1D). The inventors
further discovered that over expression of TRPV4 in TECs normalizes the
various abnormal characteristics of the TECs. For example, over
expression of TRPV4 in TECs reduced the high basal level of active Rho
(FIGS. 5A and 5B), decreased the TEC rate of migration (FIG. 3), and
decreased aberrant mechanosensory and orientation response to external
mechanic stimuli compared to TECs that were not overexpressing TRPV4
(FIG. 2). The inventors also found that the absence of TRPV4 in the
TRPV4-/- knockout mice (KO) promoted increased aberrant angiogenesis that
led to increased tumor growth in these mice (FIGS. 7 and 8).

[0006] Since the aberrant angiogenesis in tumors and abnormal TECs can be
corrected by increasing TRPV4 expression, an approach that rectifies the
TRPV4 expression deficiency in TECs can normalize angiogenesis in
cancerous situations. Alternatively, an approach that increases the cell
signaling pathway via a TRPV4 receptor in TECs can normalize angiogenesis
as well as normalize the observed aberrant characteristics described
herein. In addition, avenues for modulating the abnormal TECs
characteristics and responses such that the characteristics and responses
are closer to that of nECs can normalize angiogenesis in tumors. For
example, methods of reducing the basal level of Rho activity, decreased
the TEC rate of cell migration, and decreased aberrant mechanosensory and
orientation response to external mechanic stimuli. Furthermore, methods
of inhibiting the development of abnormal angiogenesis in a tumor can
also normalize angiogenesis having blood vessels with less vascular
leakage and networks that are closer to those formed by nECs, and this
can improve the delivery of anti-cancer therapies to the tumor and
thereby improve the efficacy of an anti-cancer therapy. Normalizing
angiogenesis in tumors can also increased sensitization to anti-cancer
therapy, e.g., radiosensitization for radiation therapy.

[0007] Accordingly, in one embodiment, provided here in is a TRPV4 agonist
or a vector comprising a DNA sequence encoding a TRVP4 for increasing the
efficacy of an anti-cancer treatment in a patient in need thereof.

[0008] In another embodiment, provided here in is a TRPV4 agonist or a
vector comprising a DNA sequence encoding a TRVP4 for the treatment of
cancer in a patient in need thereof.

[0009] In other embodiments, a TRPV4 agonist or a vector comprising a DNA
sequence encoding a TRVP4 can be used for increasing the expression of a
TRPV4 receptor in a TEC in a patient, increasing cell signaling via a
TRPV4 receptor in a TEC in a patient, modulating the abnormal
characteristics and responses of a TEC in a patient, reducing the basal
level of active Rho of a TEC, in a patient, decreasing the rate of
migration of a TEC, in a patient, decreasing aberrant mechanosensory and
orientation responses to external mechanic stimuli of a TEC in a patient,
inhibiting the development of abnormal angiogenesis in a tumor in a
patient, inhibiting tumor growth in a patient, reducing vascular leakage
in a tumor of a patient, for normalizing angiogenesis in a patient and
enhancing the radiosensitivity of a tumor to radiation therapy in a
patient.

[0010] In one embodiment, provided here in is a method for increasing the
efficacy of an anti-cancer treatment in a patient in need thereof, the
method comprising administering a TRPV4 agonist or a vector comprising a
DNA sequence encoding TRVP4 to the patient concurrently with a cancer
treatment or subsequently administering the cancer treatment to the
patient.

[0011] In one embodiment, provided here in is a method for cancer
treatment in a patient in need thereof, the method comprising
administering a TRPV4 agonist or a vector comprising a DNA sequence
encoding TRVP4 to the patient concurrently with a cancer treatment or
subsequently administering the cancer treatment to the patient.

[0012] In one embodiment of the methods described, the method further
comprises selecting a patient who has been diagnosed with cancer. In one
embodiment, the patient is diagnosed with cancer. In other embodiments,
the patient is about to start a cancer treatment or is being treated with
the cancer treatment.

[0013] In one embodiment, the TRPV4 agonist or a vector is administered
concurrently with an anti-cancer treatment or the anti-cancer treatment
is administered subsequently.

[0014] In one embodiment, the TRPV4 agonist is selected from a group
consisting of GSK1016790A, Bisandrographolide A (BAA), RN 1747,
AB1644034, α-phorbol 12,13-didecanoate (4αPDD) 5,6-EET,
acetylcholine and App441-1.

[0015] In one embodiment, the TRVP4 is a human TRVP4.

[0016] In one embodiment, the human TRVP4 is SEQ. ID. NO. 3, 4 or 5.

[0017] In one embodiment, the cancer treatment is chemotherapy, radiation
therapy and/or immunotherapy.

[0018] As used herein, "cancer" refers to any of various malignant
neoplasms characterized by the proliferation of anaplastic cells that
tend to invade surrounding tissue and metastasize to new body sites and
also refers to the pathological condition characterized by such malignant
neoplastic growths.

[0019] As used herein, "normalizes" when used in reference to a tumor
endothelial cell's characteristics such as basal level of active Rho,
endothelial cell rate of migration, and mechanosensory and orientation
response to external mechanic stimuli, vascular leakage etc refers to
characteristics that are similar or close to that of normal, non-tumor,
non-cancer derived endothelial cells (nEC) or structures form by nECs.
The use of a TRPV4 agonist or a vector comprising a DNA sequence encoding
a TRVP4 for the methods and uses described can normalize the TEC anywhere
from 5% to 100% close to that of nECs. In one embodiment, the TEC's
characteristic is normalized such that there is no difference from that
of a nEC.

[0020] In one embodiment, as used herein, "normalize angiogenesis" and
"normalizing angiogenesis" when used in reference to TECs refers to the
normal tubular network formation when the TECs are plated at high cell
density instead of the TEC forming multicellular clumps without any
tubular network formation. Normal tubular network formation occurs for
nEC when they are plated at high density. TECs exhibiting "normalized
angiogenesis" will make tubular network instead of forming multicellular
clumps. (See FIG. 6) The use of a TRPV4 agonist or a vector comprising a
DNA sequence encoding a TRVP4 for the methods and uses described can
normalize angiogenesis by TEC anywhere from 5% to 100%.

[0021] As used herein, "radiosensitization for radiation therapy" refers
to making tumors more sensitive to radiation emission such that a lower
dose of radiation is sufficient to effect more cell death in the tumor
compared to prior to radiosensitization.

[0025] FIG. 1D is a histogram showing the quantitative analysis of the
calcium influxes shown in FIG. 1C in TECs compared to normal ECs.

[0026] FIG. 2A is a histogram showing the relative calcium ion influx into
control TECs compared to NECs expressing TRPV4 from an exogenous DNA
sequence encoding TRPV4. Exogenous expression of TRPV4 increases calcium
ion influx in the tumor ECs. Control tumor ECs do not expressing TRPV4
from an exogenous DNA sequence encoding TRPV4.

[0027] FIG. 2B is a graph showing the projected cell spread area for
control TECs in response to external tension stress compared to TECs
expressing TRPV4 from an exogenous DNA sequence encoding TRPV4. Exogenous
expression of TRPV4 normalizes the responses to external tension stress
in the tumor ECs. The dotted line shows the projected cell spread area
for normal EC (nEC).

[0028] FIG. 2C contain phase contrast micrographs showing the cell spread
of control a TEC responding to external tension stress compared to the
response of a TEC expressing TRPV4 from an exogenous DNA sequence
encoding TRPV4. Exogenous expression of TRPV4 normalizes the responses to
external tension stress in the tumor ECs.

[0029] FIG. 3A shows time lapse phase contrast micrographs of a migrating
control TEC showing leading edge and trailing end of the cell. Control
tumor ECs do not expressing TRPV4 from an exogenous DNA sequence encoding
TRPV4.

[0030] FIG. 3B shows time lapse phase contrast micrographs of a migrating
TEC expressing TRPV4 from an exogenous DNA sequence encoding TRPV4. The
migrating cell shows leading edge and trailing end of the cell.

[0031] FIG. 3C is a histogram showing the cell migration rates of control
TECs compared to TECs expressing TRPV4 from an exogenous DNA sequence
encoding TRPV4. Exogenous expression of TRPV4 reduces the cell migration
rate in the TECs.

[0033] FIG. 4B is a histogram showing the percent cell migration into the
scratch zone by TECs expressing GFP (control) compared to TECs expressing
TRPV4-EGFP from an exogenous DNA sequence encoding TRPV4. Exogenous
expression of TRPV4 reduces the percent migration in the tumor ECs into
the scratch zone.

[0034] FIG. 5A shows Western blots demonstrating the reduced level of
active Rho in TECs expressing TRPV4-EGFP from an exogenous DNA sequence
encoding TRPV4 (TEC+V4) compared to control TECs that are not transfected
with the exogenous DNA sequence.

[0035] FIG. 5B is a histogram showing the relative levels of Rho activity
in TECs expressing TRPV4-EGFP from an exogenous DNA sequence encoding
TRPV4 (TEC+V4) compared to control TECs that were not transfected with
the exogenous DNA sequence.

[0036] FIG. 6A is a phase contrast micrograph showing control TECs form
multicellular cluster aggregation in an angiogenesis assay on
MATRIGEL®. The control TECs were not transfected with an exogenous DNA
sequence encoding TRPV4-EGFP.

[0037] FIG. 6B is a phase contrast micrograph of showing normalization of
tube formation by the overexpression of TRPV4 in TECs in an angiogenesis
assay on MATRIGEL®.

[0038] FIG. 7A is a graph showing that the tumor growth is enhanced in
TRPV4 knockout mice (KO) compared to control mice (WT) expressing
endogenous amounts of TRPV4. The data shown are +SEM of three independent
experiments (n=11 mice for each group).

[0039] FIG. 7B is a histogram showing that the tumor growth is enhanced in
TRPV4 knockout mice (KO) compared to control mice (WT) expressing
endogenous amounts of TRPV4.

[0042] FIG. 8B is a histogram showing that quantitative analysis of blood
microvessel density of the subcutaneously implanted tumor is increased in
TRPV4 knockout mice (KO) compared to subcutaneously implanted tumors in
control mice (WT) expressing endogenous amounts of TRPV4.

DETAILED DESCRIPTION OF THE INVENTION

[0043] Embodiments of the present invention are based on the discovery
that tumor endothelial cells (TECs) have abnormal characteristics and
responses compared to non-cancer or non-tumor associated normal
endothelial cells (nECs) Unlike nECs, TECs express abnormally low levels
of a stress-activated (SA) ion channel receptor TRPV4 compared to nECs
(FIGS. 1A and 1B). These TECs also have reduced calcium influx upon
stimulation of the TRPV4 receptor (FIGS. 1C and 1D). The inventors
further discovered that over expression of TRPV4 in TECs normalizes the
various abnormal characteristics of the TECs. For example, over
expression of TRPV4 in TECs reduced the high basal level of active Rho
(FIGS. 5A and 5B), decreased the TEC rate of migration (FIG. 3), and
decreased aberrant mechanosensory and orientation response to external
mechanic stimuli compared to TECs that were not overexpressing TRPV4
(FIG. 2). The inventors also found that the absence of TRPV4 in the
TRPV4-/- knockout mice (KO) promoted increased aberrant angiogenesis that
led to increased tumor growth in these mice (FIGS. 7 and 8).

[0044] Since the aberrant angiogenesis in tumors and abnormal TECs can be
corrected by increasing TRPV4 expression, an approach that rectifies the
TRPV4 expression deficiency in TECs can normalize angiogenesis in
cancerous situations. Alternatively, an approach that increases the cell
signaling pathway via a TRPV4 receptor in TECs can normalize angiogenesis
as well as normalize the observed abberent characteristics described
herein. In addition, avenues for modulating the abnormal TECs
characteristics and responses such that the characteristics and responses
are closer to that of nECs can normalize angiogenesis in tumors. For
example, methods of reducing the basal level of Rho activity, decreased
the TEC rate of cell migration, and decreased aberrant mechanosensory and
orientation response to external mechanic stimuli. Furthermore, methods
of inhibiting the development of abnormal angiogenesis in a tumor can
also normalize angiogenesis having blood vessels with less vascular
leakage and networks that are closer to those formed by nECs, and this
can improve the delivery of anti-cancer therapies to the tumor and
thereby improve the efficacy of an anti-cancer therapy. Normalizing
angiogenesis in tumors can also increased sensitization to anti-cancer
therapy, e.g., radiosensitization for radiation therapy.

[0045] Accordingly, provided herein is a method of improving the efficacy
of an anti-cancer therapy by normalizing angiogenesis in cancer
situations, e.g., in a patient having cancer. By enhancing a TRPV4 cell
signaling pathway in TECs, either by a TRPV4 agonist or by increasing the
expression of TRPV4 in the TECs, the TECs exhibit less abnormal
endothelial characteristics and form angiogenic network that are closer
to the networks of nECs for a more effective delivery of anti-cancer
therapy to the tumors.

[0046] In one embodiment, provided herein is a method for increasing the
efficacy of an anti-cancer treatment in a patient in need thereof, the
method comprises administering a TRPV4 agonist or a vector comprising a
DNA sequence encoding TRPV4 to the patient concurrently with a cancer
treatment or subsequently administering the cancer treatment to the
patient. In one embodiment, the patient is diagnosed with cancer. In
other embodiments, the patient is about to start a cancer treatment or is
being treated with the cancer treatment. In one embodiment, the increased
in efficacy of the anti-cancer treatment in the patient is at least 5%
compared to a control reference. In some embodiments, the increased in
efficacy is at least 10%, at least 15%, at least 20%, at least 25%, at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at
least 100% compared to the control reference, including all the
percentages to the second decimal place between 5%-100%.

[0047] In one embodiment, provided herein is a method for increasing the
expression of a TRPV4 receptor in a TEC in a patient in need thereof, the
method comprises administering a vector comprising a DNA sequence
encoding a TRPV4 to the patient. In one embodiment, the vector is
administered concurrently with a cancer treatment or the cancer treatment
is administered subsequently to the patient after the vector. In one
embodiment, the patient is diagnosed with cancer. In other embodiments,
the patient is about to start a cancer treatment or is being treated with
the cancer treatment. In one embodiment, the increased in expression of
TRPV4 is at least 5% compared a control reference. In some embodiments,
the increased in TRPV4 expression in TECs is at least 10%, at least 15%,
at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 99%, or at least 100% of the control reference,
including all the percentages to the second decimal places between
5-100%.

[0048] In another embodiment, provided herein is a method for increasing
cell signaling via a TRPV4 receptor in a TEC in a patient in need
thereof, the method comprises administering a TRPV4 agonist or a vector
comprising a DNA sequence encoding a TRPV4 to the patient. In one
embodiment, the TRPV4 agonist or vector is administered concurrently with
a cancer treatment or the cancer treatment is administered subsequently
to the patient after the TRPV4 agonist. In other embodiments, the patient
is about to start a cancer treatment or is being treated with the cancer
treatment. In one embodiment, the increased in cell signaling is measured
as an increase in calcium influx in the TECs of a patient administered a
TRPV4 agonist or vector compared to the TECs of a control patient not
administered a TRPV4 agonist or a vector comprising a DNA sequence
encoding a TRPV4. In one embodiment, the patient is diagnosed with
cancer. In one embodiment, the increase in cell signaling, calcium influx
in the TECs of the patient administered with a TRPV4 agonist is at least
5% compared to a control reference. In some embodiments, the increase is
at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 99%, or at least 100% or
more than the control reference, including all the percentages to the
second decimal places between 5-100%.

[0049] In one embodiment, provided herein is a method for modulating the
abnormal characteristics and responses of a TEC in a patient in need
thereof, the method comprises administering a TRPV4 agonist or a vector
comprising a DNA sequence encoding TRPV4 to the patient concurrently with
a cancer treatment or subsequently administering the cancer treatment to
the patient. In one embodiment, the modulation of the TEC is towards the
normal characteristics and responses of a non-cancerous, nEC. In another
embodiment, provided herein is a method for normalizing the abnormal
characteristics and responses of a TEC in a patient in need thereof, the
method comprises administering a TRPV4 agonist or a vector comprising a
DNA sequence encoding TRPV4 to the patient concurrently with a cancer
treatment or subsequently administering the cancer treatment to the
patient. In one embodiment, the modulation of the TEC is negative whereby
the characteristics and responses of the TEC is closer to that of a
non-cancerous, nEC. For example, the modulated TEC has reduced the basal
level of Rho activity, decreased the TEC rate of migration, and decreased
aberrant mechanosensory and orientation response to external mechanic
stimuli compared to control TEC not activated by a TRPV4 agonist or a
vector comprising a DNA sequence encoding TRPV4. In some embodiments, the
characteristics and responses of TEC that is modulated are the basal
level of Rho activity, the TEC rate of migration, and the mechanosensory
and orientation response to external mechanic stimuli. In one embodiment,
the modulated TEC is at least 5% closer to a control reference. In some
embodiments, the characteristics and responses of endothelial cells are
measured in terms of basal level of active Rho, endothelial cell rate of
migration, and mechanosensory and orientation response to external
mechanic stimuli. In some embodiments, the modulated TEC is at least 10%,
at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 99%, or at least 100% closer to the
control reference, including all the percentages to the second decimal
places between 5-100%. In one embodiment, the patient is diagnosed with
cancer. In other embodiments, the patient is about to start a cancer
treatment or is being treated with the cancer treatment.

[0050] In one embodiment, provided herein is a method for reducing the
basal level of active Rho of a TEC in a patient in need thereof, the
method comprises administering a TRPV4 agonist or a vector comprising a
DNA sequence encoding TRPV4 to the patient concurrently with a cancer
treatment or subsequently administering the cancer treatment to the
patient. In another embodiment, provided herein is a method for
normalizing the basal level of active Rho of a TEC, in a patient in need
thereof, the method comprises administering a TRPV4 agonist or a vector
comprising a DNA sequence encoding TRPV4 to the patient concurrently with
a cancer treatment or subsequently administering the cancer treatment to
the patient. In one embodiment, the reduction or normalization is such
that the basal level of Rho activity in the TEC is at least 5% closer to
a control reference. In one embodiment, the control reference is the
basal level of Rho activity in nECs. In some embodiments, the reduction
or normalization of Rho activity in TECs is at least 10%, at least 15%,
at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 99%, or at least 100% closer to the control
reference, including all the percentages to the second decimal places
between 5-100%. In one embodiment, the patient is diagnosed with cancer.
In other embodiments, the patient is about to start a cancer treatment or
is being treated with the cancer treatment.

[0051] In one embodiment, provided herein is a method for decreasing the
rate of migration of a TEC in a patient in need thereof, the method
comprises administering a TRPV4 agonist or a vector comprising a DNA
sequence encoding TRPV4 to the patient concurrently with a cancer
treatment or subsequently administering the cancer treatment to the
patient. In another embodiment, provided herein is a method for
normalizing the rate of migration of a TEC, in a patient in need thereof,
the method comprises administering a TRPV4 agonist or a vector comprising
a DNA sequence encoding TRPV4 to the patient concurrently with a cancer
treatment or subsequently administering the cancer treatment to the
patient. In one embodiment, the decreased or normalized rate of migration
is at least 5% closer to a control reference. In one embodiment, the
control reference is the average migrate rate of TECs in patients not
treated with a TRPV4 agonist or a vector comprising a DNA sequence
encoding TRPV4. In another embodiment, the control reference is average
cell migration rate of nECs. In some embodiments, the decreased or
normalized rate of migration is at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at
least 99%, or at least 100% closer to the control reference, including
all the percentages to the second decimal places between 5-100%. In one
embodiment, the patient is diagnosed with cancer. In other embodiments,
the patient is about to start a cancer treatment or is being treated with
the cancer treatment.

[0052] In one embodiment, provided herein is a method for decreasing
aberrant mechanosensory and orientation responses to external mechanic
stimuli of a TEC in a patient in need thereof, the method comprises
administering a TRPV4 agonist or a vector comprising a DNA sequence
encoding TRPV4 to the patient concurrently with a cancer treatment or
subsequently administering the cancer treatment to the patient. In
another embodiment, provided herein is a method for normalizing aberrant
mechanosensory and orientation responses to external mechanic stimuli of
a TEC in a patient in need thereof, the method comprises administering a
TRPV4 agonist or a vector comprising a DNA sequence encoding TRPV4 to the
patient concurrently with a cancer treatment or subsequently
administering the cancer treatment to the patient. The normalized
mechanosensory and orientation responses would be closer to those of
nECs. In one embodiment, the decreased or normalized aberrant
mechanosensory and orientation responses is at least 5% closer to a
control reference. In one embodiment, the control reference is the
average mechanosensory and orientation responses to external mechanic
stimuli of nECs. In some embodiments, the decreased or normalized
aberrant mechanosensory and orientation responses is at least 10%, at
least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at
least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95%, at least 99%, or at least 100% closer to the
control reference, including all the percentages to the second decimal
places between 5-100%. In one embodiment, the patient is diagnosed with
cancer. In other embodiments, the patient is about to start a cancer
treatment or is being treated with the cancer treatment.

[0053] In one embodiment, provided herein is a method for inhibiting the
development of abnormal angiogenesis in a tumor in a patient in need
thereof, the method comprises administering a TRPV4 agonist or a vector
comprising a DNA sequence encoding TRPV4 to the patient concurrently with
a cancer treatment or subsequently administering the cancer treatment to
the patient. In one embodiment, the method comprises contacting the tumor
with a TRPV4 agonist or a vector comprising a DNA sequence encoding
TRPV4. For example, directly injecting the TRPV4 agonist or vector into
the tumor in the patient. In one embodiment, the patient is diagnosed
with cancer. In other embodiments, the patient is about to start a cancer
treatment or is being treated with the cancer treatment. In some
embodiments, the inhibition of the development of abnormal angiogenesis
in a tumor is measured in terms of basal level of active Rho, endothelial
cell rate of migration, and mechanosensory and orientation response to
external mechanic stimuli of a TEC in the patient. For example, the TECs
of a patient administered with a TRPV4 agonist or a vector comprising a
DNA sequence encoding TRPV4 are isolated and assayed for Rho activity,
cell migration rate, and mechanosensory and orientation responses no
external stimuli. These TECs would have reduced the basal level of active
Rho, decreased the TEC rate of migration, and decreased aberrant
mechanosensory and orientation response to external mechanic stimuli
compared to the TECs of a patient to whom the TRPV4 agonist or a vector
comprising a DNA sequence encoding TRPV4 was not administered. In another
embodiment, the inhibition of the development of abnormal angiogenesis in
a tumor is assessed by an in vitro angiogenesis assay of the isolated
TECs before and after the application of the TRPV4 agonist or a vector.
These TECs would have reduced multicellular retractions and cell clumping
and increased tube formation. In another embodiment, the inhibition of
the development of abnormal angiogenesis in a tumor is measured by
imaging the network of blood vessels in the tumor before and after the
application of the TRPV4 agonist or vector. The network of blood vessels
in the tumors would be less thick with large clumps of TECs. In one
embodiment, the inhibition of the development of abnormal angiogenesis in
a tumor is inhibited by at least 5% closer compared to a control
reference. In one embodiment, the control reference is the average
abnormal angiogenesis in tumors of patients not administered the TRPV4
agonist or a vector described herein.

[0054] In some embodiments, the inhibition of the development of abnormal
angiogenesis in a tumor is inhibited by at least 10%, at least 15%, at
least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 99%, or at least 100% compared to the control
reference, including all the percentages to the second decimal places
between 5-100%.

[0055] Methods of assessing angiogenesis are known to those skilled in the
art, such as, in vitro cell migration and capillary tube formation as
described by Nicosia R. F. and Ottinetti A, (In Vitro Cell Dev. Biol.,
1990, 26:119-128), Ghosh et al., (PNAS, 2008, 105:11305-11310), Lingen
MW, (Methods Mol. Med. 2003, 78:337-47), and McGonigle and Shifrin,
(Curr. Prot. Pharmacology, 2008, Unit 12.12). Other methods include but
are not limited to dynamic contrast-enhanced MRI (DCE-MRI) which can be
used to demonstrate tissue perfusion and permeability. Moreover, MRI with
macromolecular contrast media (MMCM) can depict microvessel permeability
and fractional plasma volume. (Padhani, A. R., British Journal of
Radiology (2003) 76, S60-S80).

[0056] The level of angiogenesis and/or the network of blood vessels in
the tumors in patients can be measured by micro-CT angiography with
contrast reagents, dynamic contrast-enhanced MRI (DCE-MRI) and MRI with
macromolecular contrast media (MMCM). Examples of contrast reagents for
use with these imaging methods include by are not limited to the low
molecular weight Gd(III) contrast reagents such as gadoteridol and the
macromolecular iron oxide CRs such as ferumoxytol.

[0057] In some embodiments, commercial angiogenesis assays can be used.
For example, the MATRIGEL® assay where ECs are plated in wells coated
with MATRIGEL® (Becton Dickinson, Cedex, France). Alternatively, an in
vitro angiogenesis assay kit marketed by CHEMICON® can be used. The
Fibrin Gel In Vitro Angiogenesis Assay Kit is CHEMICON® Catalog No.
ECM630.

[0058] In one embodiment, the inhibition of abnormal angiogenesis is such
that there is at least 5% reduction in Rho activity, cell migration rate,
and/or mechanosensory and orientation responses no external stimuli
compared to nEC or at least 5% reduction in the multicellular
retractions, cell clumping and/or thickness of the blood vessels in the
tumor compared to the TECs or network of blood vessels before the
application of the TRPV4 agonist or vector. In some embodiments, the
inhibition of abnormal angiogenesis is at least 10%, at least 15%, at
least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at
least 95%, at least 99%, or at least 100% reduction in Rho activity, cell
migration rate, and mechanosensory and orientation responses no external
stimuli compared to nEC orreduction in the multicellular retractions,
cell clumping and/or thickness of the blood vessels in the tumor compared
to the TECs or network of blood vessels before the application of the
TRPV4 agonist or vector, including all the percentages to the second
decimal places between 5-100%.

[0059] The inventors found that the absence of TRPV4 in the TRPV4-/-
knockout mice promoted increased aberrant angiogenesis in tumors which
led to increased tumor growth in these mice. While not wishing to be
bound by theory, increased TRPV4 expression or cell signaling in TECs via
TRPV4 can inhibit aberrant angiogenesis which can lead to the inhibition
of tumor growth.

[0060] In one embodiment, provided herein is a method for inhibiting tumor
growth in a patient in need thereof, the method comprises administering a
TRPV4 agonist or a vector comprising a DNA sequence encoding TRPV4 to the
patient concurrently with a cancer treatment or subsequently
administering the cancer treatment to the patient. In one embodiment, the
method comprises contacting the tumor with a TRPV4 agonist or a vector
comprising a DNA sequence encoding TRPV4. For example, directly injecting
the TRPV4 agonist or the vector into the tumor in the patient. In one
embodiment, the patient is diagnosed with cancer. In other embodiments,
the patient is about to start a cancer treatment or is being treated with
the cancer treatment. In one embodiment, the tumor growth is reduced by
at least 5% compared to the tumor size prior to administration of the
TRPV4 agonist or vector. In some embodiments, the tumor growth is reduced
by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 99%, or at least 100%
compared to the tumor size prior to administration of the TRPV4 agonist
or vector, including all the percentages to the second decimal places
between 5-100%. In one embodiment, the inhibition is complete absence or
disappearance of the tumor by currently detection method. Methods of
measuring the size of a tumor in a patient are well known to a skill
clinician, physician or oncologist. For example, MRI, CAT scanning (CT),
X-ray, mammography and 18F-FDG PET scans.

[0061] In another embodiment, provided herein is a method for treatment of
cancer in a patient in need thereof, the method comprises administering a
TRPV4 agonist or a vector comprising a DNA sequence encoding TRPV4 to the
patient concurrently with a cancer treatment or subsequently
administering the cancer treatment to the patient. In one embodiment, the
method comprises contacting the tumor with a TRPV4 agonist or a vector
comprising a DNA sequence encoding TRPV4. For example, directly injecting
the TRPV4 agonist or the vector into the tumor in the patient. In one
embodiment, the patient is diagnosed with cancer. In other embodiments,
the patient is about to start a cancer treatment or is being treated with
the cancer treatment. Efficiacy of the treatment can be determined by any
methods that are known in the art and those described herein.

[0062] In another embodiment, provided herein is a method for reducing
vascular leakage in a tumor of a patient in need thereof, the method
comprises administering a TRPV4 agonist or a vector comprising a DNA
sequence encoding TRPV4 to the patient concurrently with a cancer
treatment or subsequently administering the cancer treatment to the
patient. In one embodiment, the method comprises contacting the tumor
with a TRPV4 agonist or a vector comprising a DNA sequence encoding
TRPV4. For example, directly injecting the TRPV4 agonist or the vector
into the tumor in the patient. In one embodiment, the patient is
diagnosed with cancer. In other embodiments, the patient is about to
start a cancer treatment or is being treated with the cancer treatment.
While not wishing to be bound by theory, increased TRPV4 expression or
cell signaling in TECs can normalized TECs' abnormal characteristics then
can lead to the formation of blood vessels that are less hyperpermeable
and les internal pressure. In one embodiment, the vascular leakage is
reduced by at least 5% in the tumor compared to the leakage prior to
administration of the TRPV4 agonist or vector. In another embodiment, the
vascular leakage is reduced by at least 5% in the tumor compared to a
control reference. In some embodiments, the vascular leakage is reduced
by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at
least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at least 90%, at least 95%, at least 99%, or at least 100%
prior to administration of the TRPV4 agonist or vector, or compared to
the control reference, including all the percentages to the second
decimal places between 5-100%. Methods of assessing vascular permeability
are known to those ordinary skilled in the art. For example, using a
hyperpolarized 1H-MRI, known as Overhauser enhanced MRI (OMRI) and
an oxygen-sensitive contrast agent OX63 as described in Matsumotoa S. et
al., (PNAS, 2009, 106: 17898-17903), by DCE-MRI or by using FITC dextran
and multiphoton fluorescence intravital microscopy as described in
Reyes-Aldasoro, C. C., et al. (Angiogenesis, 2006, 9:26), by
14C-iodoantipyrine (IAP)-quantitative autoradiography (QAR)
(IAP-QAR) as described in Ewing J R, et al., (J. Cereb. Blood Flow Metab.
2003, 23:198-209) and by Evans blue dye extrusion as described by van der
Heyde, H. C. et al. (Infection & Immunity, 2001, 69: 3460-3465).

[0063] In another embodiment, provided herein is a method for enhancing
radiosensitivity to radiation therapy in a patient in need thereof, the
method comprises administering a TRPV4 agonist or a vector comprising a
DNA sequence encoding TRPV4 to the patient concurrently with a cancer
treatment or subsequently administering the cancer treatment to the
patient. In one embodiment, the method comprises contacting the tumor
with a TRPV4 agonist or a vector comprising a DNA sequence encoding
TRPV4. For example, directly injecting the TRPV4 agonist or the vector
into the tumor in the patient. In one embodiment, the patient is
diagnosed with cancer. In other embodiments, the patient is about to
start a cancer treatment or is being treated with the cancer treatment.
In one embodiment, the cancer treatment is radiation therapy. In one
embodiment, the radiosensitivity of a tumor of the same type to radiation
therapy is enhanced by at least 5% compared to the radiosensitivity of
the tumor prior to administration of the TRPV4 agonist or vector. In one
embodiment, the radiosensitivity of a tumor to radiation therapy is
enhanced by at least 5% compared to a control reference. In one
embodiment, the control reference is the average data of radiosensitivity
of tumors from a control population of patients not administration of the
TRPV4 agonist or vector. In some embodiments, the radiosensitivity is
enhanced by at least 10%, at least 15%, at least 20%, at least 25%, at
least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at
least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at
least 100% prior to administration of the TRPV4 agonist or vector, or
compared to a control reference, including all the percentages to the
second decimal places between 5-100%. Radiosensitivity of tumors can be
assessed by any method known in the art. e.g., by assessing the amount of
cell death before and after radiation therapy.

[0064] The inventors previously isolated TECs from prostrate
adenocarinoma, studied various characteristics and responses of these
TECs and shown that the TECs were very different from non-tumor derived,
normal endothelial cells. TECs exhibited defective strain-induced
reorientation of the cell main axis and actin cytoskeleton, exhibited
abnormal mechano sensitivity to substrate elasticity compared to nECs by
way of enhanced ability to spread to any given substrate elasticity,
exhibited enhanced readiness to form capillary networks in vitro when
plated at low cell density but not at high cell density, exhibited
multicellular retraction, cell clumping and no capillary network
formation in vitro when plated at very high cell density, and also
exerted stronger Rho-mediated traction on their extracellular matrix
adhesions (Ghosh et al., 2008, PNAS, 105:11305-11310). In response to
uniaxial cyclic strain, nECs would re-orientate the cell main axis and
actin cytoskeleton perpendicular to the direction of the strain (Iba and
Sumpio, 1991, Microvasc. Res. 42:245-254; Ghosh et al., 2008, PNAS,
105:11305-11310). In contrast, under uniaxial cyclic strain, the TECs do
not re-orientate the cell main axis and actin cytoskeleton perpendicular
to the direction of the strain. This failure can be as much as 40% of the
time.

[0065] Recently, the inventors discovered that TECs had reduced levels of
TRPV4 expression and reduced calcium influx upon TRPV4 stimulation
compared to nECs (FIG. 1). The TRPV4 expression level in TECs was reduced
by 10% to 75% (FIG. 1B). The calcium influx was reduced by 10% to 50%
(FIG. 1C). More interestingly, the inventors discovered that by
increasing TRPV4 expression in the TECs, the abnormal characteristic were
reduced, normalized and/or restored to be closer to that of nECs. For
example, TRPV4 expression restored the abnormal mechanosensitivity to
substrate elasticity, inhibited the enhanced rate of cell migration (FIG.
3), e.g., in a scratch wound assay (FIG. 4), inhibited the abnormal basal
Rho activity (FIG. 5), and promoted capillary networks formation in vitro
instead of multicellular retraction and cell clumping when the TECs were
plated at very high cell density in an in vitro angiogenesis assay (FIG.
6). For example, the TECs migration rate was reduced by as much as 90%
compared to control TECs that did not exhibit increased TRPV4 expression
(FIG. 3C). The abnormal basal Rho activity was reduced by about 60%
compared to control TECs that did not exhibit increased TRPV4 expression
(FIG. 5A).

[0066] This discovery that increasing TRPV4 expression in the TECs
inhibited abnormal angiogenesis and normalizes angiogenesis by these TECs
in vitro was surprising. This is because previously TRPV4 was identified
as an important contributor to the ability of normal endothelial cells to
sense and respond to mechanical stress that is necessary for the
formation of new blood vessels (see WO 2009/149239). TRPV4 is a major
mechanochemical "transducer" of mechanical strain in nEC. TRPV4
transduces the strain into a chemical signal intracellularly through the
activation of β1 integrin, a transmembrane protein receptor that
links the cytoskeleton to the extracellular matrix. It is known that
mechanical strains influence the re-arrangement of the cells'
cytoskeleton which in turn affects the migration capability of normal EC
that is needed for re-aligning and/or reorienting the cells during
angiogenesis. Therefore, inhibition of TRPV4 expression or the downstream
cell signaling pathway is useful for inhibiting nEC cell alignment, nEC
cell migration, capillary tube formation and overall angiogenesis (see WO
2009/149239). The discovery that increasing TRPV4 expression in the TECs
inhibited abnormal angiogenesis is the exact opposite of the current
teachings of TRPV4 in relationship to angiogenesis.

[0067] In one embodiment of the described methods, the control reference
is the data obtained for a control population of patients all having the
same type of cancer and were being treated or would be treated with the
same anti-cancer therapy but were not administered with a TRPV4 agonist
or a vector comprising a DNA sequence encoding TRPV4 concurrently or
subsequently with the cancer treatment. In other words, the control
population of patients was cancer type-matched and anti-cancer
therapy-matched with the patient treated with TRPV4 agonist or vector.

[0069] In one embodiment of the described methods, the data is the average
level of TRPV4 expression in TECs from the control population of patients
that are not administered a vector comprising a DNA sequence encoding a
TRPV4. The level of TRPV4 expression can be measured by methods well
known in the art, for example, quantitative reverse transcription
polymerase chain reaction (qRT-PCR) with specific primers or by measuring
the amount of the protein TRPV4, e.g., Western blot analysis. Exemplary
primer pair for qRT-PCT of the human TRPV4 transcript is the forward
primer GACGGGGACCTATAGCATCA (SEQ. ID. NO. 1) and the reverse primer
AACAGGTCCAGGAGGAAGGT (SEQ. ID. NO. 2). Exemplary commercially available
TRPV4 antibodies of Western blot analysis are catalog No: ab62992 from
ABCAM, catalog No: AB9334-SOUL and catalog No: AB9336-200UL from
Millipore, and catalog No: LS-C95115 and catalog No: LS-C95200 from
Lifespan Bioscience Inc.

[0070] In one embodiment of the described methods, the data of the control
population is the average increase in calcium influx in the TEC of
patients that were not administered a TRPV4 agonist. In this embodiment,
the cell signaling is measured by an increase in calcium influx in the
TECs of a patient administered a TRPV4 agonist compared to the TECs of a
patient not administered a TRPV4 agonist. Methods of monitoring calcium
in cell are well known in the art. For example, by flow cytometry (June,
C. H., et al., Current Protocols in Cytometry, unit 9.8, 2001) and by
fluorescence Spectroscopy of calcium sensitive dyes, e.g., Fura-2, Indo-1
oregon green bapta-1, Fluo-4 and Fluo-3. Alternatively, commercially
available Fluo-4 Direct® Calcium Assay Kit by INVITROGEN® and The
Wash free Fluo-8 Calcium Assay kits by HD Biosciences Co., Ltd can be
used.

[0071] An exemplary method of measurement of intracellular calcium influx
is provided as follows. TECs were isolated from tumor biopsy from
patients that were administered with a TRPV4 agonist and from patients
(control) not given a TRPV4 agonist. The TECs were mixed in culture with
Fura-2 AM, rinse of free Fura-2 AM and then transferred to a quartz
cuvette and the fluorescence measured at excitation wavelengths of 340
and 380 nm and an emission wavelength of 510 nm (LS50B Luminescence
Spectrometer; Perkin Elmer, Buckinghamshire, UK). During the fluorescence
measurements the cells were maintained in suspension using a magnetic
stirrer and the cuvette was thermostatically controlled at 37° C.
The ratio of the fluorescence values at excitation wavelengths of 340 and
380 nm were calibrated and converted to Ca2+ concentration (nM)
according to the protocol of Grynkiewicz et al. (J Biol Chem 1985; 260:
3440-3450) as follows.

[ Ca 2 + ] c = Kd ( R - R min ) ( R
max - R ) ##EQU00001##

[0072] Kd is 224 nM, the apparent dissociation constant for Ca2+ and
Fura-2. The maximum ratio (Rmax) was obtained by the addition of Triton
X-100 (0.5%) to lyse the cells. The minimum ratio (Rmin) was obtained by
the addition of EGTA (7 mM, added as a 0.5 M stock buffered with 3M
tris-hydroxymethyl-amino methase (Tris)-HCl).

[0073] In one embodiment of the described methods, the data of the control
population is the average characteristics and responses of non-tumor or
non-cancer associated endothelial cells. In some embodiments, the
characteristics and responses of the endothelial cells are measured in
terms of basal level of Rho activity, the rate of cell migration in
vitro, and/or the mechanosensory and orientation response to external
mechanic stimuli. In one embodiment, the data of the control population
is the average basal level of Rho activity in a population of nEC. In
another embodiment, the data of the control population is the average
rate of cell migration in vitro for a population of nECs. In another
embodiment, the data of the control population is the average
mechanosensory and orientation responses to external mechanic stimuli for
a population of nECs. In one embodiment, the nECs are obtained from
patients administered with a TRPV4 agonist or a vector comprising a DNA
sequence encoding TRPV4. In another embodiment, the nECs are obtained
from patients that are not administered with a TRPV4 agonist or a vector
comprising a DNA sequence encoding TRPV4.

[0075] In one embodiment, the data of the control population is the
average radiosensitivity of tumors from patients not administration of
the TRPV4 agonist or vector. Methods of assessing tumor radiosensitivity
are known to those skilled in the art, e.g., physician, oncologist etc.
For example, tumor radiosensitivity can be monitored by metabolic
functional imaging using positron emission tomography (PET) as described
in Belkacemi Y., et al. (Crit Rev Oncol Hematol. 2007, 62:227-39), by
assessing amount of cell death, by using an in vitro a soft agar
clonogenic assay of biopsy sample to a single dose of 2 Gy radiation
(SF2) as described in Wilson C R., et al. (British J. Cancer,
200083:1702-1706) or by short-term proliferative assays such as
[3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
reduction, methylene blue staining, and [3H]-thymidine incorporation] as
described in Peter Cross M.D. et al. (Radiation Oncology Investigations,
1993, 1:261-269).

[0076] In one embodiment, the number of patients in the control population
can range from 5-2000. In one embodiment, the patients in the control
population also have the same stage of cancer, i.e. they are cancer
stage-matched with the TRPV4 agonist or a vector treated patient. In one
embodiment, the patients in the control population are also aged-matched
within an age range, i.e. they are age-matched with the TRPV4 agonist or
a vector treated patient. In one embodiment, the patients in the control
population are gender-matched. Therefore, they are of the same gender as
the TRPV4 agonist or a vector treated patient. In one embodiment, the
patients in the control population are also race-matched, e.g.,
Caucasians, African, Hispanic, Asian etc, i.e. the patients in the
control population are of the same or similar race as the TRPV4 agonist
or a vector treated patient.

[0077] In one embodiment of the methods described, the method further
comprises selecting a patient who has been diagnosed with cancer. As used
herein, "cancer" refers to any of various malignant neoplasms
characterized by the proliferation of anaplastic cells that tend to
invade surrounding tissue and metastasize to new body sites and also
refers to the pathological condition characterized by such malignant
neoplastic growths.

[0078] In one embodiment of the methods described, the patient is
diagnosed with cancer. Methods of diagnosing cancer are known to a
skilled physician. In general, cancer is suspected based on a person's
symptoms, the results of a physical examination, and the results of
screening tests such as imaging. Imaging tests often include plain
x-rays, ultrasonography, CT, and MRI. These tests assist in identifying
abnormalities, determining qualities of a mass (solid or cystic),
providing dimensions, and establishing relationship to surrounding
structures, which can be important if surgery or biopsy is being
considered. Occasionally, x-rays obtained for other reasons such as an
injury, show abnormalities that might be cancer. Confirmation that cancer
is present requires other tests (termed diagnostic tests e.g, by tumor
biopsy and histopathologic examination). Other screening tests include
but are not limited to screening the level of serum tumor markers the
findings of which are suggestive of a specific cancer. For examples
α-Fetoprotein (hepatocellular carcinoma, testicular carcinoma),
carcinoembryonic antigen (colon cancer), β-human chorionic
gonadotropin (choriocarcinoma, testicular carcinoma), serum
immunoglobulins (multiple myeloma), DNA probes (eg, bcr probe to identify
a chromosome 22 alteration in chronic myelogenous leukemia), CA 125
(ovarian cancer), CA 27-29 (breast cancer), prostate-specific antigen
(prostate cancer).

[0079] After cancer is diagnosed, it is staged. Staging is a way of
describing how extensive or advanced the cancer is in terms of its
location, size, growth into nearby structures, and spread to other parts
of the body. People with cancer sometimes become impatient and anxious
during staging tests, wishing for a prompt start of treatment. However,
staging allows doctors to determine the most appropriate treatment as
well as helping to determine prognosis.

[0080] Staging may use scans or other imaging tests, such as x-ray, CT,
MRI, bone scintigraphy, or positron emission tomography (PET). The choice
of staging test(s) depends on the type of cancer, as different cancers
involve different parts of the body. CT scanning is used to detect cancer
in many parts of the body, including the brain and lungs and parts of the
abdomen, including the adrenal glands, lymph nodes, liver, and spleen.
MRI is of particular value in detecting cancers of the brain, bone, and
spinal cord.

[0081] Biopsies are often needed for staging and can sometimes be done
together with the initial surgical treatment of a cancer. For example,
during a laparotomy (an abdominal operation) to remove colon cancer, a
surgeon removes nearby lymph nodes to check for spread of the cancer.
During surgery for breast cancer, the surgeon biopsies or removes lymph
nodes located in the armpit to determine whether the breast cancer has
spread there; this information along with features of the primary tumor
helps the doctor determine whether further treatment is needed. When
staging is based only on initial biopsy results, physical examination,
and imaging, the stage is referred to as clinical. When the doctor uses
results of a surgical procedure or additional biopsies, the stage is
referred to as pathologic. The clinical and pathologic stage may differ

[0082] In addition to imaging tests, doctors often obtain blood tests to
see if the cancer has begun to affect the liver, bone, or kidneys.

[0084] In one embodiment of the methods described, the cancer treatment is
chemotherapy. Chemotherapy treatment uses medicine to weaken and destroy
cancer cells in the body, including cells at the original cancer site and
any cancer cells that may have spread to another part of the body.
Chemotherapy can also aims at keeping the cells from further multiplying.
The majority of chemotherapeutic drugs can be divided in to alkylating
agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase
inhibitors, and other antitumour agents. All of these drugs affect cell
division or DNA synthesis and function in some way. Examples of
chemotherapeutic agents include but are not limited to drugs such as
daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,
idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, epirubicin,
cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,
actinomycin D, mithramycin, prednisone, hydroxyprogesterone,
testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,
pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,
methylcyclohexylnitrosurea, nitrogen mustards, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),
5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,
vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,
topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol
(DES).

[0085] In one embodiment of the methods described, the cancer treatment is
immunotherapy. The principle behind cancer immunotherapy is to use of the
immune system to reject cancer. Since the immune system responds to the
environmental factors it encounters on the basis of discrimination
between self and non-self, many kinds of tumor cells that arise as a
result of the onset of cancer are more or less tolerated by the patient's
own immune system since the tumor cells are essentially the patient's own
cells that are growing, dividing and spreading without proper regulatory
control. The main premise is stimulating the patient's immune system to
attack the malignant tumor cells that are responsible for the disease.
This can be either through immunization of the patient (e.g., by
administering a cancer vaccine, such as Dendreon's Provenge), in which
case the patient's own immune system is trained to recognize tumor cells
as targets to be destroyed, or through the administration of therapeutic
antibodies as drugs, in which case the patient's immune system is
recruited to destroy tumor cells by the therapeutic antibodies.

[0086] In some embodiments, cancer immunotherapy includes but not limited
to cell-based immunotherapy, monoclonal antibody therapy, and
radioimmunotherapy.

[0087] In one embodiment of the methods described, the cancer treatment is
cell based immunotherapy. In another embodiment of the methods described,
the cancer treatment is autologous immune enhancement therapy (AIET).
Cell based immunotherapy is a major form of cancer immunotherapy. This
involves immune cells such as the natural killer cells (NK cells),
lymphokine activated killer cell (LAK), cytotoxic T lymphocytes (CTLs),
dendritic Cells (DC) etc which are either activated in vivo by
administering certain cytokines such as interleukins or they are
isolated, enriched and transfused to the patient to fight against cancer.
Cell based immunotherapy encompasses AIET which involves isolation of
either allogenic or autologous immune cells, enriching them outside the
body and transfusing them back to the patient. The injected immune cells
are highly cytotoxic to the cancer cells thereby helping to fight the
cancer cells. AIET therapy is in routine clinical practice in some
countries such as Japan.

[0088] In one embodiment of the methods described, the cancer treatment is
monoclonal antibody therapy. Monoclonal antibody therapy involves raising
antibodies against specific antigens such as the unusual antigens that
are presented on the surfaces of tumors. Many kinds of tumor cells
display unusual antigens that are either inappropriate for the cell type
and/or its environment, or are only normally present during the
organisms' development (e.g., fetal antigens). Examples of such antigens
include the glycosphingolipid GD2, a disialoganglioside that is normally
only expressed at a significant level on the outer surface membranes of
neuronal cells, where its exposure to the immune system is limited by the
blood-brain barrier. GD2 is expressed on the surfaces of a wide range of
tumor cells including neuroblastoma, medulloblastomas, astrocytomas,
melanomas, small-cell lung cancer, osteosarcomas and other soft tissue
sarcomas. GD2 is thus a convenient tumor-specific target for
immunotherapies. Other cancer antigens include but are not limited to
CD52 for chronic lymphocytic leukemia, vascular endothelial growth factor
for colorectal cancer, epidermal growth factor receptor for colorectal
cancer, CD33 for acute myelogenous leukemia, CD20 for non-Hodgkin
lymphoma, and ErbB2 for breast cancer. Anti-cancer monoclonal antibodies
include but are not limited to alemtuzumab, bevacizumab, cetuximab,
gemtuzumab ozogamicin, rituximab and trastuzumab.

[0089] In one embodiment of the methods described, the cancer treatment is
radioimmunotherapy. Radioimmunotherapy involves the use of radioactively
conjugated murine antibodies against cellular antigens, especially the
cell surface antigens that are expressed unusually described herein,
e.g., over expression, inappropriate expression temporarily and
spacially. Some kinds of tumor cells display cell surface receptors that
are rare or absent on the surfaces of healthy cells, and which are
responsible for activating cellular signal transduction pathways that
cause the unregulated growth and division of the tumor cell. Examples
include ErbB2, a constitutively active cell surface receptor that is
produced at abnormally high levels on the surface of breast cancer tumor
cells. Most radioimmunotherapy currently involved their application to
lymphomas, as these are highly radio-sensitive malignancies. To limit
radiation exposure, murine antibodies were especially chosen, as their
high immunogenicity promotes rapid clearance from the body. The two most
common are ibritumomab tiuxetan and the tositumomab/iodine (131I)
tositumomab regimen. Ibritumomab tiuxetan is a murine antibody chemically
linked to a chelating agent which binds yttrium-90. 90Y is a beta
radiator, has a half-life of 64 h and a tissue penetration of 1-5
millimetres. Its use has been investigated, primarily in the treatment of
follicular lymphoma. Tositumomab is a murine IgG2a anti-CD20 antibody.
Iodine (131I) tositumomab is covalently bound to Iodine 131.
131I emits both beta and gamma radiation, and is broken down rapidly
in the body. Tositumomab and iodine (131I) tositumomab are used in
patients with relapsed follicular lymphoma.

[0090] In one embodiment of the methods described, the cancer treatment is
radiation. Radiation therapy uses high-energy radiation to shrink tumors
and kill cancer cells. The high-energy radiation kills cancer cells by
damaging their DNA such that the cancer cells cannot multiply. X-rays,
gamma rays, and charged particles are types of radiation used for cancer
treatment. The radiation may be delivered by a machine outside the body
(external-beam radiation therapy), or it may come from radioactive
material placed in the body near cancer cells (internal radiation
therapy, also called brachytherapy). Systemic radiation therapy uses
radioactive substances, such as radioactive iodine, that travel in the
blood to kill cancer cells, e.g., thyroid cancer. Image-guided radiation
is a recent development in radiation therapy where it provides real-time
imaging of the tumor target during treatment. Real-time imaging could
help compensate for normal movement of the internal organs from breathing
and for changes in tumor size during treatment.

[0091] In some embodiments of the methods described, the radiation is
applied in conjunction with radiosensitizers and radioprotectors,
chemicals that modify a cell's response to radiation. Radiosensitizers
are drugs that make cancer cells more sensitive to the effects of
radiation therapy. Several agents are under study as radiosensitizers. In
addition, some anticancer drugs, such as 5-fluorouracil and cisplatin,
make cancer cells more sensitive to radiation therapy. Radioprotectors
(also called radioprotectants) are drugs that protect normal cells from
damage caused by radiation therapy. These drugs promote the repair of
normal cells exposed to radiation. Many agents are currently being
studied as potential radioprotectors.

[0092] In one embodiment of the methods described, the cancer treatment is
a combination of chemotherapy, immunotherapy and/or radiation therapy. In
another embodiment, the cancer treatment includes radiosensitizers and/or
radioprotectors

[0093] In one embodiment of the methods described, the TRPV4 agonist is
selected from a group consisting of GSK1016790A, Bisandrographolide A
(BAA), RN 1747, AB1644034, α-phorbol 12,13-didecanoate
(4αPDD) 5,6-EET, acetylcholine and App441-1. BAA is the active
compound from the extracts of Andrographis paniculata (Chinese herbal
plant).

[0094] In one embodiment of the methods described, the TRPV4 is a human
TRPV4. TRPV4 is a member of the TRP channels comprising a large family of
cation channels that provide a pathway for calcium influx into cells.
Among the ˜30 TRP-channel proteins identified in mammals,
endothelial cells express ˜20 members that are classified into six
subfamilies: canonical (TRPC), vanilloid (TRPV), melastatin (TRPVM),
polycystin (TRPP), mucolipin (TRPML) and TRPA. Structurally, TRP channels
consist of six transmembrane (TM)-spanning helices with a pore region
between TM5 and cytoplasmic N and C termini. Both TRPC and TRPV channels
contain multiple anykyrin domains at their N-terminus that are absent in
TRPM channels. Most of the TRP channels contain PDZ binding motifs and
recognition sites for PKC and PI3K. TRPC subfamily channels that are
ubiquitously expressed in endothelial cells are responsible for
store-operated or receptor-mediated calcium entry; they also have been
implicated in control of endothelial barrier function and vasorelaxation.
Among the vertebrate TRPV and TRPM channels, TRPV4 and TRPV2 are
considered mechanosensitive, and growing evidence suggests that TRPV4
plays critical role in mechanical force-induced regulation of endothelial
cell function. For example, in endothelial cells, TRPV4 acts as a calcium
entry channel that is activated by increases in cell volume and
temperature. TRPV4 can also be activated by ligands such as arachidonic
acid and its metabolites, endocannabinoids and a synthetic phorbol ester,
4-α-phorbol 12,13-didecanoate (PPD), and it can be suppressed by
integrin and Src kinase inhibitors during osmotransduction in dorsal root
ganglia.

[0095] The human TRPV4 gene is located on chromosome 12, location:
12q24.1, 108,705,277-108,755,595 reverse strand (ENSG00000111199)
(Ensembl) assembled in Accession No. NC--000012.10 (SEQ. ID. No. 2;
GENBANK®) Alternate gene names are OTRPC4, TRP12, VR-OAC, VRL-2, VRL2
and VROAC. This gene encodes a member of the OSM9-like transient receptor
potential channel (OTRPC) subfamily in the transient receptor potential
(TRP) superfamily of ion channels. The encoded protein is a
Ca2+-permeable, nonselective cation channel that is thought to be
involved in the regulation of systemic osmotic pressure. Two transcript
variants encoding different isoforms have been found for this gene. Two
transcripts of TRPV4 from this gene are NM--021625.3 (SEQ. ID. No.
4) and NM--147204.1 (SEQ. ID. No. 5) (GENBANK®).

[0096] In one embodiment of the methods described, the DNA sequence that
encodes a TRPV4 comprises SEQ. ID. NO. 4 or 5, the two messenger
transcript variants of the human TRPV4. In another embodiment of the
methods described, the DNA sequence that encodes a TRPV4 consists
essentially of SEQ. ID. NO. 4 or 5, the two messenger transcript variants
of the human TRPV4. In another embodiment of the methods described, the
DNA sequence that encodes a TRPV4 consists of SEQ. ID. NO. 4 or 5, the
two messenger transcript variants of the human TRPV4.

[0097] In one embodiment of the methods described, the DNA sequence that
encodes a TRPV4 comprises the genomic sequence 108,705,277-108,755,595
reverse strand on chromosome 12, location: 12q24.1 (SEQ. ID. No. 2). In
another embodiment of the methods described, the DNA sequence that
encodes a TRPV4 consisting essentially of SEQ. ID. No. 2. In another
embodiment of the methods described, the DNA sequence that encodes a
TRPV4 consists essentially of SEQ. ID. No. 2. In another embodiment of
the methods described, the DNA sequence that encodes a TRPV4 consists of
SEQ. ID. No. 2.

[0098] In one embodiment of the methods described, the DNA sequence that
encodes a TRPV4 is in a vector. In one embodiment, the vector is an
expression vector for the purpose of expressing a DNA sequence encoding a
protein in a cell. In one embodiment, the vector is an inducible vector,
such as a tetracycline inducible vector. Methods described, for example,
in Wang et al. Proc. Natl. Acad. Sci. 100: 5103-5106, using pTet-On
vectors (BD Biosciences Clontech, Palo Alto, Calif.) can be used. In some
embodiments, a vector is a plasmid vector, a viral vector, or any other
suitable vehicle adapted for the insertion and foreign sequence and for
the introduction into eukaryotic cells. The vector can be an expression
vector capable of directing the transcription of the DNA sequence
enroding TRPV4.

[0099] In one embodiment, the expression vector is a viral vector. Viral
expression vectors can be selected from a group comprising, for example,
reteroviruses, lentiviruses, Epstein Barr virus-, bovine papilloma virus,
adenovirus- and adeno-associated-based vectors or hybrid virus of any of
the above. In one embodiment, the vector is episomal. The use of a
suitable episomal vector provides a means of maintaining the antagonist
nucleic acid molecule in the subject in high copy number extra
chromosomal DNA thereby eliminating potential effects of chromosomal
integration.

[0100] Any methods known in the art can be for constructing a vector for
the purpose of expressing a DNA sequence encoding a TRPV4 in a cell. For
example, conventional polymerase chain reaction (PCR) cloning techniques
can be used to clone the DNA sequence encoding a TRPV4. A DNA sequence
encoding a TRPV4 can be initially cloned into a general purpose cloning
vector such as pUC19, pBR322, pBluescript vectors (STRATAGENE® Inc.)
or pCR TOPO® from INVITROGEN® Inc. prior to cloning into the
expression vector.

[0101] Each PCR primer should have at least 15 nucleotides overlapping
with its corresponding templates at the region to be amplified. The
polymerase used in the PCR amplification should have high fidelity such
as STRATAGENE®'s PFUULTRA® polymerase for reducing sequence
mistakes during the PCR amplification process. For ease of ligating
several separate PCR fragments together, for example in the construction
of a genomic DNA sequence encoding TRPV4 such as SEQ. ID. NO: 2, and
subsequently inserting into a cloning vector, the PCR primers should also
have distinct and unique restriction digestion sites on their flanking
ends that do not anneal to the DNA template during PCR amplification. The
choice of the restriction digestion sites for each pair of specific
primers should be such that the DNA sequence encoding a TRPV4 is in-frame
and will encode the predicted TRPV4 protein from beginning to end with no
stop codons.

[0102] In gene therapy, a vector comprising a DNA sequence encoding a
TRPV4 includes but is not limited to adenovirus, retrovirus, lentivirus,
adeno associated virus, envelope protein pseudotype virus (chimeric
virus), and virosomes (e.g. liposomes combined with an inactivated HIV or
influenza virus).

[0104] Recombinant lentivirus has the advantage of delivery and expression
of a TRPV4 in either dividing or non-dividing mammalian cells. The HIV-1
based lentivirus can effectively transduce a broader host range than the
Moloney Leukemia Virus (MoMLV)-base retroviral systems. Preparation of
the recombinant lentivirus can be achieved using the pLenti4/V5-DEST®,
pLenti6/V5-DEST® or pLenti vectors together with ViraPower®
Lentiviral Expression systems from INVITROGEN®.

[0105] An embodiment is the use of AAV viral vectors comprising nucleic
acids encoding a TRPV4. Recombinant adeno-associated virus (rAAV) vectors
are applicable to a wide range of host cells including many different
human and non-human cell lines or tissues. Because AAV is non-pathogenic
and does not ellicit an immune response, a multitude of pre-clinical
studies have reported excellent safety profiles. rAAVs are capable of
transducing a broad range of cell types and transduction is not dependent
on active host cell division. High titers, >108 viral
particle/ml, are easily obtained in the supernatant and 1011-1012 viral
particle/ml with further concentration. The transgene is integrated into
the host genome so expression is long term and stable.

[0107] Large scale preparation of AAV vectors is made by a three-plasmid
cotransfection of a packaging cell line: AAV vector carrying the coding
nucleic acid, AAV RC vector containing AAV rep and cap genes, and
adenovirus helper plasmid pDF6, into 50×150 mm plates of
sub-confluent 293 cells. Cells are harvested three days after
transfection, and viruses are released by three freeze-thaw cycles or by
sonication.

[0108] AAV vectors are then purified by two different methods depending on
the serotype of the vector. AAV2 vector is purified by the single-step
gravity-flow column purification method based on its affinity for heparin
(Auricchio, A., et. al., 2001, Human Gene therapy 12:71-6; Summerford, C.
and R. Samulski, 1998, J. Virol. 72:1438-45; Summerford, C. and R.
Samulski, 1999, Nat. Med. 5: 587-88). AAV2/1 and AAV2/5 vectors are
currently purified by three sequential CsCl gradients.

[0109] Formulation and Administration

[0110] In one embodiment of the methods described, the method comprises
administering a composition comprising a TRPV4 agonist or a vector
comprising a DNA sequence encoding a TRPV4 and a pharmaceutically
acceptable carrier.

[0111] In one embodiment, the composition further comprises a polymer. In
one embodiment, the polymer comprises block co-polymers.

[0112] In one embodiment of the composition, the polymer forms
nanoparticles.

[0113] In another embodiment, the composition further comprises a
targeting agent. For example, to target the TRPV4 agonist or vector
comprising a DNA sequence encoding a TRPV4 to the cancer site is a
targeted delivery vehicle, e.g., a liposome, microparticle or
nanoparticle. Specially targeted delivery vehicles can function to
increase effective levels of the TRPV4 agonist or vector comprising a DNA
sequence encoding a TRPV4 for tumor cells while reducing effective levels
for other cells. This should result in an increased tumor kill and/or
reduced toxicity. In general, specially targeted delivery vehicles have a
differentially higher affinity for tumor cells by interacting with
tumor-specific or tumor-associated antigens.

[0114] Specially targeted delivery vehicles vary in their stability,
selectivity, and choice of target, but, in essence, they all aim to
increase the maximum effective dose that can be delivered to the tumor
cells. Reduced systemic toxicity means that they can also be used in
sicker patients, and that they can carry new chemotherapeutic agents that
would have been far too toxic to deliver via traditional systemic
approaches.

[0115] In one embodiment, the targeting agent enhances accumulation of the
composition or components within in a solid tumor. The TRPV4 agonist,
vector comprising a DNA sequence encoding a TRPV4 or composition
comprising there of can be targeted to specific organ or tissue by means
of a targeting moiety, such as e.g., an antibody or targeted liposome
technology. In some embodiments, targeting to tissue- or tumor-specific
targets is by using bispecific antibodies, for example produced by
chemical linkage of an anti-ligand antibody (Ab) and an Ab directed
toward a specific target. To avoid the limitations of chemical
conjugates, molecular conjugates of antibodies can be used for production
of recombinant bispecific single-chain Abs directing ligands and/or
chimeric inhibitors at cell surface molecules. The conjugation of the
TRPV4 agonist, vector comprising a DNA sequence encoding a TRPV4 or
composition comprising thereof permits the TRPV4 agonist or vector
comprising a DNA sequence encoding a TRPV4 to attached and to accumulate
additively at the desired target site. Antibody-based or
non-antibody-based targeting moieties can be employed to deliver the
TRPV4 agonist or vector comprising a DNA sequence encoding a TRPV4 to a
target site. Preferably, a natural binding agent for an unregulated or
disease associated antigen is used for this purpose. For example, Albumin
is playing an increasing role as a drug carrier in the clinical setting.
This is because there is substantial accumulation of albumin in solid
tumors and this fact forms the rationale for developing albumin-based
drug delivery systems for tumor targeting. A methotrexate-albumin
conjugate, an albumin-binding prodrug of doxorubicin, i.e. the
(6-maleimido)caproylhydrazone derivative of doxorubicin (DOXO-EMCH), and
an albumin paclitaxel nanoparticle (Abraxane) have been evaluated
clinically. Abraxane has been approved for treating metastatic breast
cancer. Albuferon, a fusion protein of albumin and interferon is
currently being assessed. (Kratz F. J., Control Release. 2008,
18:132(3):171-83). Partly PEGylated polyamidoamine (PAMAM) dendrimers
were used as the carrier for tumor-selective targeting of the anticancer
drug doxorubicin (DOX). Acid-sensitive cis-aconityl linkage or
acid-insensitive succinic linkage was introduced between DOX and
polymeric carriers to produce PPCD or PPSD conjugates, respectively. DOX
release from PPCD conjugates followed an acid-triggered manner and
increased with increasing PEGylation degree. In vitro cytotoxicity of
PPCD conjugates against murine B16 melanoma cells increased with, while
cellular uptake decreased with increasing PEGylation degree. (Zhu S. et
al, Biomaterials, 2010, 31:1360-71).

[0116] In some embodiments, the targeting agent is covalently or
non-covalently linked to the polymer. In another embodiment, the
targeting agent is covalently or non-covalently linked to the TRPV4
agonist or vector. Methods of linking are well known in the art, e.g., a
bi-functional linker described in WO 2007/034479, Mei H., et al.,
Biomaterials. 2010, 31:5619-26, Hu K, en al., J Control Release. 2009,
134:55-61, Chen Z., et al., J Drug Target. 2010 Nov. 23; and Santosh
Aryal, ACS Nano, 2010, 4:251-258.

[0117] In one embodiment, the composition further comprises a cancer
therapeutic agent. In one embodiment of the composition, the cancer
therapeutic agent is for chemotherapy, radiotherapy or immunotherapy.

[0118] In one embodiment of the methods described, the TRPV4 agonist or
the vector comprising a DNA sequence encoding a TRPV4 is delivered with
or in a pharmaceutically acceptable carrier.

[0119] In one embodiment, the term "pharmaceutically acceptable" means
approved by a regulatory agency of the Federal or a state government or
listed in the U.S. Pharmacopeia or other generally recognized
pharmacopeia for use in animals, and more particularly in humans.
Specifically, it refers to those compounds, materials, compositions,
and/or dosage forms which are, within the scope of sound medical
judgment, suitable for use in contact with the tissues of human beings
and animals without excessive toxicity, irritation, allergic response, or
other problem or complication, commensurate with a reasonable
benefit/risk ratio.

[0120] The term "carrier" refers to a diluent, adjuvant, excipient, or
vehicle with which the therapeutic is administered. Such pharmaceutical
carriers can be sterile liquids, such as water and oils, including those
of petroleum, animal, vegetable or synthetic origin, such as peanut oil,
soybean oil, mineral oil, sesame oil and the like. Water is a preferred
carrier when the pharmaceutical composition is administered
intravenously. Saline solutions and aqueous dextrose and glycerol
solutions can also be employed as liquid carriers, particularly for
injectable solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, water, ethanol and the like. The
composition, if desired, can also contain minor amounts of wetting or
emulsifying agents, or pH buffering agents. These compositions can take
the form of solutions, suspensions, emulsion, tablets, pills, capsules,
powders, sustained-release formulations, and the like. The composition
can be formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard carriers
such as pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
Examples of suitable pharmaceutical carriers are described in Remington's
Pharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing Co.,
1990). The formulation should suit the mode of administration. Additional
carrier agents, such as liposomes, can be added to the pharmaceutically
acceptable carrier.

[0121] As used herein, the terms "administering," refers to the placement
of a TRPV4 agonist, a vector comprising a DNA sequence encoding a TRPV4
or a composition comprising the TRPV4 agonist or the vector comprising a
DNA sequence encoding a TRPV4 into a patient by a method or route which
results in at least partial localization of the TRPV4 at a desired site.
The TRPV4 agonist, vector comprising a DNA sequence encoding a TRPV4 or
composition can be administered by any appropriate route which results in
an effective treatment in the patient.

[0122] As used herein, the term "comprising" or "comprises" is used in
reference to methods, and respective component(s) thereof, that are
essential to the invention, yet open to the inclusion of unspecified
elements, whether essential or not. The use of "comprising" indicates
inclusion rather than limitation.

[0123] The term "consisting of" refers to methods, and respective
components thereof as described herein, which are exclusive of any
element not recited in that description of the embodiment.

[0124] Compositions that are therapeutic for the methods described herein
contain a physiologically tolerable carrier together with an active agent
as described herein, dissolved or dispersed therein as an active
ingredient. The active ingredient is a TRPV4 agonist or a vector
comprising a DNA sequence encoding a TRPV4. The active ingredient can
include more that one TRPV4 agonist, e.g., a mixture of two, three, or up
to five TRPV4 agonists. In a preferred embodiment, the therapeutic
composition is not immunogenic when administered to a mammal or human
patient for therapeutic purposes. As used herein, the terms
"pharmaceutically acceptable", "physiologically tolerable" and
grammatical variations thereof, as they refer to compositions, carriers,
diluents and reagents, are used interchangeably and represent that the
materials are capable of administration to or upon a mammal without the
production of undesirable physiological effects such as nausea,
dizziness, gastric upset and the like. A pharmaceutically acceptable
carrier will not promote the raising of an immune response to an agent
with which it is admixed, unless so desired. The preparation of a
pharmacological composition that contains active ingredients dissolved or
dispersed therein is well understood in the art and need not be limited
based on formulation. Typically such compositions are prepared as
injectable either as liquid solutions or suspensions, however, solid
forms suitable for solution, or suspensions, in liquid prior to use can
also be prepared. The preparation can also be emulsified or presented as
a liposome composition. The active ingredient can be mixed with
excipients which are pharmaceutically acceptable and compatible with the
active ingredient and in amounts suitable for use in the methods
described herein. Specifically contemplated pharmaceutical compositions
include those comprising a TRPV4 agonist or a vector comprising a DNA
sequence encoding a TRPV4 in a preparation for delivery as described
herein above, or in references cited and incorporated herein in that
section. Suitable excipients include, for example, water, saline,
dextrose, glycerol, ethanol or the like and combinations thereof. In
addition, if desired, the composition can contain minor amounts of
auxiliary substances such as wetting or emulsifying agents, pH buffering
agents and the like which enhance the effectiveness of the active
ingredient. The therapeutic composition for the methods described herein
can include pharmaceutically acceptable salts of the components therein.
Pharmaceutically acceptable salts include the acid addition salts (formed
with the free amino groups of the polypeptide) that are formed with
inorganic acids such as, for example, hydrochloric or phosphoric acids,
or such organic acids as acetic, tartaric, mandelic and the like. Salts
formed with the free carboxyl groups can also be derived from inorganic
bases such as, for example, sodium, potassium, ammonium, calcium or
ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.
Physiologically tolerable carriers are well known in the art. Exemplary
liquid carriers are sterile aqueous solutions that contain no materials
in addition to the active ingredients and water, or contain a buffer such
as sodium phosphate at physiological pH value, physiological saline or
both, such as phosphate-buffered saline. Still further, aqueous carriers
can contain more than one buffer salt, as well as salts such as sodium
and potassium chlorides, dextrose, polyethylene glycol and other solutes.
Liquid compositions can also contain liquid phases in addition to and to
the exclusion of water. Exemplary of such additional liquid phases are
glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
The amount of an active agent used in the methods described herein that
will be effective in the treatment of a particular disorder or condition
will depend on the nature of the disorder or condition, and can be
determined by standard clinical techniques.

[0125] The method of delivering the composition comprising a TRPV4 agonist
or a vector comprising a DNA sequence encoding a TRPV4 will vary based on
the individual patient, the type and location of cancer being treated and
other criteria evident to one of ordinary skill in the art. Delivery
methods include direct injection at the treatment site, percutaneous
delivery for injection, percutaneous delivery for topical application,
and other delivery methods well known to persons of ordinary skill in the
art.

[0127] Topical administration of a pharmacologically effective amount may
utilize transdermal delivery systems well known in the art. An example is
a dermal patch. Topical and transdermal delivery can be accomplished via
a wound dressing impregnated with a TRPV4 agonist or a vector comprising
a DNA sequence encoding a TRPV4 enter the dermis and also enter the blood
stream. Alternatively the biolistic gene gun method of delivery may be
used. The gene gun is a device for injecting cells with genetic
information, originally designed for plant transformation. The payload is
an elemental particle of a heavy metal coated with plasmid DNA. This
technique is often simply referred to as biolistics. Another instrument
that uses biolistics technology is the PDS-1000/He particle delivery
system. The vector comprising a DNA sequence encoding a TRPV4 can be
coated on minute gold particles, and these coated particles are "shot"
into cancer tissues such as and melanoma under high pressure. An example
of the gene gun-based method is described for DNA based vaccination of
cattle by Loehr B. I. et al. J. Virol. 2000, 74:6077-86. Other direct
injection delivery methods, including intramuscular, intracoronary and
subcutaneous injections, can be accomplished using a needle and syringe,
using a high pressure, needle free technique, like POWDERJECT®,
constant infusion pump, a catheter delivery system, or the injection
apparati disclosed in the International Patent Publication number WO
2007112136.

[0128] In addition to topical administration, the TRPV4 agonist, the
vector comprising a DNA sequence encoding a TRPV4 or the composition
comprising thereof described herein can also be administered systemically
in a pharmaceutical formulation. For example, the TRPV4 agonist, the
vector comprising a DNA sequence encoding a TRPV4 or the composition
comprising thereof can be administered intravenously, e.g. via central
venous catheter (CVC or central venous line or central venous access
catheter) placed into a large vein in the neck (internal jugular vein),
chest (subclavian vein) or groin (femoral vein).

[0129] Systemic routes include but are not limited to oral, parenteral,
nasal inhalation, intratracheal, intrathecal, intracranial, and
intrarectal. The pharmaceutical formulation is preferably a sterile
saline or lactated Ringer's solution. For therapeutic applications, the
preparations described herein are administered to a human, in a
pharmaceutically acceptable dosage form, including those that may be
administered to a human intravenously as a bolus or by continuous
infusion over a period of time, by intramuscular, intraperitoneal,
intracerebrospinal, subcutaneous, intra-arterial, intrasynovial,
intrathecal, oral, topical, or inhalation routes. The TRPV4 agonist, the
vector comprising a DNA sequence encoding a TRPV4 or the composition
comprising thereof described herein are also suitably administered by
intratumoral, peritumoral, intralesional or perilesional routes, to exert
local as well as systemic effects. The intraperitoneal route is expected
to be particularly useful, for example, in the treatment of ovarian
tumors. For these uses, additional conventional pharmaceutical
preparations such as tablets, granules, powders, capsules, and sprays may
be preferentially required. In such formulations further conventional
additives such as binding-agents, wetting agents, propellants,
lubricants, and stabilizers may also be required. In one embodiment, the
therapeutic compositions described herein are formulated in a cationic
liposome formulation such as those described for intratracheal gene
therapy treatment of early lung cancer (Zou Y. et. al., Cancer Gene Ther.
2000 May; 7(5):683-96). The liposome formulations are especially suitable
for aerosol use in lung cancer patients. Vector DNA and/or virus can be
entrapped in `stabilized plasmid-lipid particles` (SPLP) containing the
fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10
mol %) of cationic lipid, and stabilized by a polyethyleneglycol (PEG)
coating (Zhang Y. P. et. al. Gene Ther. 1999, 6:1438-47). Other
techniques in formulating expression vectors and virus as therapeutics
are found in "DNA-Pharmaceuticals: Formulation and Delivery in Gene
Therapy, DNA Vaccination and Immunotherapy" by Martin Schleef (Editor)
December 2005, Wiley Publisher, and "Plasmids for Therapy and
Vaccination" by Martin Schleef (Editor) May 2001, are incorporated herein
as reference. In one embodiment, the dosage for viral vectors is
1×106 to 1×1014 viral vector particles per
application per patient.

[0130] The TRPV4 agonist, the vector comprising a DNA sequence encoding a
TRPV4 or the composition comprising thereof can be formulated as a
sustained release composition of formulation. For example,
sustained-release pharmaceutical compositions include, but are not
limited to, sustained-release matrices such as biodegradable matrices or
semi-permeable polymer matrices in the form of shaped articles, e.g.,
films, or mirocapsules that comprise the TRPV4 agonist, the vector
comprising a DNA sequence encoding a TRPV4 or the composition comprising
thereof.

[0131] A sustained-release matrix, as used herein, is a matrix made of
materials, usually polymers, which are degradable by enzymatic or
acid/base hydrolysis or by dissolution. Once inserted into the body, the
matrix is acted upon by enzymes and body fluids. The sustained-release
matrix desirably is chosen from biocompatible materials such as
liposomes, polylactides (polylactic acid), polyglycolide (polymer of
glycolic acid), polylactide co-glycolide (co-polymers of lactic acid and
glycolic acid)polyanhydrides, poly(ortho)esters, polyproteins, hyaluronic
acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids,
phospholipids, polysaccharides, nucleic acids, polyamino acids, amino
acids such as phenylalanine, tyrosine, isoleucine, polynucleotides,
polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferred
biodegradable matrix is a matrix of one of polylactide, polyglycolide, or
polylactide co-glycolide (co-polymers of lactic acid and glycolic acid).

[0133] For examples of sustained release compositions, see U.S. Pat. No.
3,773,919, EP 58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian
Patent No. 1176565, U. Sidman et al., Biopolymers 22:547 (1983) and R.
Langer et al., Chem. Tech. 12:98 (1982). The TRPV4 agonist or the
composition comprising thereof described herein will usually be
formulated in such vehicles at a concentration of about 0.1 mg/ml to 100
mg/ml and the vector comprising a DNA sequence encoding a TRPV4 should be
in the range of 1×106 to 1×1014 viral vector
particles per application per patient.

[0134] In one embodiment, osmotic minipumps are used to provide controlled
sustained delivery of pharmaceutical compositions described herein,
through cannulae to the site of interest, e.g. directly into a tissue at
the site of metastatic growth or into the vascular supply of a tumor. The
pump can be surgically implanted, for example continuous administration
of endostatin, an anti-angiogenesis agent, by intraperitoneally implanted
osmotic pump is described in Cancer Res. 2001 Oct. 15; 61(20):7669-74.
Therapeutic amounts of the TRPV4 agonist, the vector comprising a DNA
sequence encoding a TRPV4 or the composition comprising thereof can also
be continually administered by an external pump attached to an
intravenous needle.

[0135] For enteral administration, a composition can be incorporated into
an inert carrier in discrete units such as capsules, cachets, tablets or
lozenges, each containing a predetermined amount of the active compound;
as a powder or granules; or a suspension or solution in an aqueous liquid
or non-aqueous liquid, e.g., a syrup, an elixir, an emulsion or a
draught. Suitable carriers may be starches or sugars and include
lubricants, flavorings, binders, and other materials of the same nature.

[0136] A tablet may be made by compression or molding, optionally with one
or more accessory ingredients. Compressed tablets may be prepared by
compressing in a suitable machine the active compound in a free-flowing
form, e.g., a powder or granules, optionally mixed with accessory
ingredients, e.g., binders, lubricants, inert diluents, surface active or
dispersing agents. Molded tablets may be made by molding in a suitable
machine, a mixture of the powdered active compound with any suitable
carrier.

[0137] A syrup or suspension may be made by adding the active compound to
a concentrated, aqueous solution of a sugar, e.g., sucrose, to which may
also be added any accessory ingredients. Such accessory ingredients may
include flavoring, an agent to retard crystallization of the sugar or an
agent to increase the solubility of any other ingredient, e.g., as a
polyhydric alcohol, for example, glycerol or sorbitol.

[0138] Formulations for rectal administration may be presented as a
suppository with a conventional carrier, e.g., cocoa butter or Witepsol
S55 (trademark of Dynamite Nobel Chemical, Germany), for a suppository
base.

[0140] Alternatively, the TRPV4 agonist, the vector comprising a DNA
sequence encoding a TRPV4 or the composition comprising thereof can be
administered in liposomes or microspheres (or microparticles). Methods
for preparing liposomes and microspheres for administration to a patient
are well known to those of skill in the art. U.S. Pat. No. 4,789,734, the
contents of which are hereby incorporated by reference, describes methods
for encapsulating biological materials in liposomes. A review of known
methods is provided by G. Gregoriadis, Chapter 14, "Liposomes," Drug
Carriers in Biology and Medicine, pp. 287-341 (Academic Press, 1979).

[0141] Microspheres formed of polymers or proteins are well known to those
skilled in the art, and can be tailored for passage through the
gastrointestinal tract directly into the blood stream. Alternatively, the
compound can be incorporated in the microspheres, or composite of
microspheres, implanted for slow release over a period of time ranging
from days to months. See, for example, U.S. Pat. Nos. 4,906,474,
4,925,673 and 3,625,214, and Jein, TIPS 19:155-157 (1998), the contents
of which are hereby incorporated by reference.

[0142] Preferred microparticles are those prepared from biodegradable
polymers, such as polyglycolide, polylactide and copolymers thereof.
Those of skill in the art can readily determine an appropriate carrier
system depending on various factors, including the desired rate of drug
release and the desired dosage.

[0143] In one embodiment, the formulations are administered via catheter
directly to the inside of blood vessels. The administration can occur,
for example, through holes in the catheter. The formulations comprising
the TRPV4 agonist, the vector comprising a DNA sequence encoding a TRPV4
or the composition comprising thereof can be included in biodegradable
polymeric hydrogels, such as those disclosed in U.S. Pat. No. 5,410,016
to Hubbell et al. These polymeric hydrogels can be delivered to the
inside of a tissue lumen and the active compounds released over time as
the polymer degrades. If desirable, the polymeric hydrogels can have
microparticles or liposomes which include the active compound dispersed
therein, providing another mechanism for the controlled release of the
active compounds.

[0144] The precise dose and formulation to be employed depends upon the
potency of the TRPV4 agonist or the vector comprising a DNA sequence
encoding a TRPV4, and include amounts large enough to produce the desired
effect, e.g., an increased cell signaling via the TRPV4 receptor and/or
by an increased expression of TRPV4 in the tumor ECs. The dosage should
not be so large as to cause unacceptable adverse side effects. Generally,
the dosage will vary with the type of TRPV4 agonist or vector, and with
the age, condition, and sex of the patient are also considered. Dosage
and formulation of the TRPV4 agonist, the vector comprising a DNA
sequence encoding a TRPV4 or the composition comprising thereof will also
depend on the route of administration, and the type, stage, location of
cancer, and should be decided according to the judgment of the
practitioner and each patient's circumstances. Effective doses can be
extrapolated from dose-response curves derived from in vitro or animal
model test systems.

[0146] Administration of the doses recited above can be repeated for a
limited period of time. In some embodiments, the doses are given once a
day, or multiple times a day, for example but not limited to three times
a day. In a preferred embodiment, the doses recited above are
administered daily for several weeks or months. The duration of treatment
depends upon the subject's clinical progress and responsiveness to
therapy. Continuous, relatively low maintenance doses are contemplated
after an initial higher therapeutic dose.

[0147] As exemplary, for the treatment of solid tumors that are accessible
by catheters or needles, the TRPV4 agonist or the vector comprising a DNA
sequence encoding a TRPV4 and a pharmaceutically acceptable carrier can
be formulated for direct application by injection into the solid tumor
and/or adjacent to the tumor site, e.g., melanoma. The TRPV4 agonist or
the vector comprising a DNA sequence encoding a TRPV4 can also be
formulated for a transdermal delivery, e.g. a skin patch. For cancers or
tumors not so easily accessible, the TRPV4 agonist or the vector
comprising a DNA sequence encoding a TRPV4 can be administered to one of
the main blood vessel that drains the cancer site, e.g. into the hepatic
portal vein for liver cancer.

[0149] Efficacy testing can be performed during the course of treatment
using the methods described herein. Measurements of the degree of
severity of a number of symptoms associated with a particular ailment are
noted prior to the start of a treatment and then at later specific time
period after the start of the treatment. Other methods of efficacy
testing include evaluating for rate of vessel growth, angiogenesis, etc.:
(1) inhibiting, arresting, or slowing the pathogenic growth of abnormal
blood vessels and irregular or abnormal angiogenesis, thickness of blood
vessel, vessel leakage in tumors; or (2) reducing the tumor growth; and
(3) preventing or reducing the angiogenesis in tumors).

[0150] Unless otherwise explained, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this disclosure belongs. Definitions of common
terms in molecular biology may be found in Benjamin Lewin, Genes IX,
published by Jones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634);
Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published
by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the singular.

[0152] It should be understood that this invention is not limited to the
particular methodology, protocols, and reagents, etc., described herein
and as such may vary. The terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to limit the
scope of the present invention, which is defined solely by the claims.

[0153] Other than in the operating examples, or where otherwise indicated,
all numbers expressing quantities of ingredients or reaction conditions
used herein should be understood as modified in all instances by the term
"about." The term "about" when used in connection with percentages will
mean±1%.

[0154] All patents and publications identified are expressly incorporated
herein by reference for the purpose of describing and disclosing, for
example, the methodologies described in such publications that might be
used in connection with the present invention. These publications are
provided solely for their disclosure prior to the filing date of the
present application. Nothing in this regard should be construed as an
admission that the inventors are not entitled to antedate such disclosure
by virtue of prior invention or for any other reason. All statements as
to the date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or contents
of these documents.

[0155] The present invention can be defined in any of the following
alphabetized paragraphs:

[0156] [A] A TRPV4 agonist or a vector
comprising a DNA sequence encoding a TRVP4 for increasing the efficacy of
an anti-cancer treatment in a patient in need thereof.

[0157] [B] A TRPV4
agonist or a vector comprising a DNA sequence encoding a TRVP4 for
treatment of cancer in a patient in need thereof.

[0158] [C] The use of
paragraph [A] or [B], wherein the TRPV4 agonist or a vector is
administered concurrently with an anti-cancer treatment or the
anti-cancer treatment is administered subsequently.

[0159] [D] The use of
paragraph [A], [B] or [C], wherein the TRPV4 agonist is selected from a
group consisting of GSK1016790A, Bisandrographolide A (BAA), RN 1747,
AB1644034, α-phorbol 12,13-didecanoate (4αPDD) 5,6-EET,
acetylcholine and App441-1.

[0160] [E] The use of paragraph [A], [B], [C]
or [D], wherein the TRVP4 is a human TRVP4.

[0161] [F] The use of
paragraph [E], wherein the human TRVP4 is SEQ. ID. NO. 3, 4 or 5.

[0162]
[G] The use of any one of paragraphs [A]-[F], wherein the cancer
treatment is chemotherapy, radiation therapy or immunotherapy.

[0163] [H]
A method for increasing the efficacy of an anti-cancer treatment in a
patient in need thereof, the method comprising administering a TRPV4
agonist or a vector comprising a DNA sequence encoding TRVP4 to the
patient concurrently with a cancer treatment or subsequently
administering the cancer treatment to the patient.

[0164] [I] A method
for cancer treatment in a patient in need thereof, the method comprising
administering a TRPV4 agonist or a vector comprising a DNA sequence
encoding TRVP4 to the patient concurrently with a cancer treatment or
subsequently administering the cancer treatment to the patient.

[0165]
[J] The method of paragraph [H] or [I], wherein the cancer treatment is
chemotherapy, radiation therapy or immunotherapy.

[0166] [K] The method
of paragraph [H], [I] or [J], wherein the TRPV4 agonist is selected from
a group consisting of GSK1016790A, Bisandrographolide A (BAA), RN 1747,
AB1644034, α-phorbol 12,13-didecanoate (4αPDD) 5,6-EET,
acetylcholine and App441-1

[0167] [L] The method of paragraph [H], [I],
[J] or [K], wherein the TRVP4 is a human TRVP4.

[0169] This invention is further illustrated by the following example
which should not be construed as limiting. The contents of all references
cited throughout this application, as well as the figures and table are
incorporated herein by reference.

[0170] Those skilled in the art will recognize, or be able to ascertain
using not more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. Such equivalents
are intended to be encompassed by the following claims.

EXAMPLE

Materials and Methods

[0171] Cell Culture. Tumor endothelial cells (EC) cells were isolated from
transgenic TRAMP mice bearing prostate adenocarcinoma. Because it is
difficult to obtain sufficient quantities of EC cells from the normal
mouse prostate (due to its small size), normal EC that were isolated from
the dermis (MDEC cells) of TRAMP mice were used instead. Normal EC cells
from human dermis (HDEC cells; Cambrex) and an established mouse
pancreatic EC cell line (MS1-EC cells; gift of Judah Folkman) served as
independent non-tumor EC cell controls. Tumor EC cells, MDEC, and MS1-EC
cells were cultured on Fibronectin-coated tissue culture dishes and grown
in culture medium composed of low glucose DMEM, 10% FBS, 10% Nu Serum IV,
basic fibroblast growth factor (6 ng/ml), heparin salt (0.1 mg/ml), 1%
insulin-transferrin-selenium, and an antibiotic/mycotic mixture. These
cells were used between passages 10-19. HDEC cells were grown on tissue
culture dishes in medium as per manufacturer's protocol and used between
passages 4-8.

Mechanical Strain Application

[0172] EC cells cultured on fibronectin-coated 6 well UNIFLEX®
(FLEXCELL® International) plates for 24 hours to 70-80% confluence
and then were subjected to uniaxial cyclic stretch (10% elongation; 1 Hz
frequency) for 18 h using a FLEXERCELL® TENSION PLUS® System
(FLEXCELL®International). In some experiments, the EC cells were
plated on fibronectin-coated 6 well BIOFLEX® (FLEXCELL®
International) for 1 h and subjected to static stretch (15% elongation)
for 1-15 min. Control cells were maintained under identical conditions in
the absence of strain application.

[0174] Transglutaminase-cross-linked gelatin hydrogels of increasing
stiffness were prepared with a final gelatin concentration of 3, 5, or
10% (wt/vol) and incubated at 37° C. overnight to stabilize
cross-linking Stiffness measurements were performed by using an AR-G2
rheometer (TA Instruments) with a standard aluminum parallel plate
geometry of 20 mm. Hydrogels were subjected to a stress sweep, and their
storage moduli (G') were compared under the same physical conditions. To
analyze the effects of varying ECM elasticity on cell shape, we cultured
EC cells for 6 h on hydrogels of varying stiffness at a low density
(1,000 cells per squared centimeter) to minimize cell-cell interactions.

Morphological and Immunofluorescence Studies

[0175] Cells adherent to flexible ECM substrates and subjected to
mechanical stretch were washed in PBS, fixed in 4% paraformaldehyde for
30 min either mounted on glass slides (for visualizing GFP-AKT-PH
translocation) or permeabilized with 0.25% TRITON®-X100/PBS for 5 min
for immuno staining. After blocking with DMEM containing 10% FBS, cells
were incubated for 1 h with ALEXA®-phalloidin to visualize stress
fibers, washed and mounted on glass slides using FLUOROMOUNT-G®
(Southern Biotech). Images were acquired on a LEICA® Confocal SP2
microscope and processed using LEICA® software and Adobe Photoshop.

[0176] EC cell reorientation in response to cyclic strain was measured by
quantitating the angle of orientation of cells relative to the direction
of applied strain using ImageJ software and MICROSOFT® EXCEL®.
Cells on substrates exposed to uniaxial cyclic strain with their longest
axis oriented between 60 and 120 degrees relative to the direction of the
applied strain field were considered to be aligned. Statistical
differences between experimental groups were determined using the student
t-test.

Biochemical Analysis

[0177] Western blotting analyses were performed according to methods
published in Mammoto et. al. (2007) (J. Cell Sci., 120:456-467).
Membranes containing transferred protein were blocked in 3% BSA/TBST for
1 h and incubated overnight with primary antibodies against TRPV4
(1:1000) at 4° C. The membranes were subsequently washed incubated
with HRP-conjugated secondary antibodies (1:5000) for 1 h and washed and
incubated with SUPERSIGNAL® West Pico ECL reagent from Pierce
Biotechnology Inc. (USA) and exposed to Kodak X-ray film (SIGMA
ALDRICH®).

In Vitro Angiogenesis Assay

[0178] Capillary network formation by EC cells was analyzed by using a
two-dimensional fibrin gel assay, which was modified from the well known
fibrin-based in vitro assay. Thrombin-crosslinked fibrin gels (3 mg/ml)
were formed in 48-well plates and incubated at 37° C. for 30 min
before normal and tumor EC cells were plated in culture medium at
densities of 2, 3, 4, or 8×104 cells per well. Cells were
cultured at 37° C. for 24 h before tube formation was analyzed. In
some experiments, cells plated at the highest density (8×104
cells per well) and cultured for 3 h were treated with Y27632 (10 μM),
and capillary network formation was monitored after 24 h. To analyze
capillary organization by EC cells cultured within (as opposed to on top
of) 3D ECM gels, normal or tumor EC cells were resuspended at a high
density (5×106 cells per milliliter) in either fibrin gel (5
mg/ml) or MATRIGEL® and cultured the cells in regular growth medium
for 1 day or 2 wk, respectively.

Cell Migration

[0179] Cell migration assay was performed using Transwell assay. Briefly,
cells were plated on to gelatin coated (0.5%) transwell membranes
(Coster) in EBM2 supplemented with 0.3% FBS and their migration in
response to VEGF (10 ng/ml) was monitored. The migrated cells were
stained with Giemsa solution for 16 h and ten random fields were counted.
To measure in vitro angiogenesis, EC cells were plated on MATRIGEL®
(BD Biosciences) and incubated in the presence of VEGF (10 ng/ml) at
37° C. After 18 h, tube formation was assessed in ten random
fields (Mamotto, 2009, Nature 457:1103-1109).

[0181] Rho activity was determined by using the Rhotekin-RBD binding assay
(Cytoskeleton). Cells grown on FN-coated flexible silicone substrates
with or without 10% uniaxial cyclic strain for 2 h were lysed in RIPA
buffer, and equal volumes of clarified lysate were treated with
GST-Rhotekin-RBD beads for 1 h at 4° C. The beads were pelleted,
washed, and treated with SDS-sample buffer to solubilized bead-bound
GTP-Rho, which was detected by using western blot analysis.

Microscopy, Image Analysis, and Statistics

[0182] Images of live cells forming tubular structures in the in vitro
angiogenesis assay and of cells cultured on compliant gelatin hydrogels
fixed with 4% paraformaldehyde were recorded by using a Nikon Diaphot 300
phase contrast microscope (Nikon) fitted with a Hamamatsu digital camera
(Hamamatsu Photonics). In other studies, cells were fixed with 4%
paraformaldehyde, permeabilized with 0.2% Triton X-100, stained with
Alexa Fluor-488 Phalloidin and DAPI (to visualize actin and nuclei,
respectively), and imaged by using an Nikon Eclipse TE 2000-E microscope
(Nikon) fitted with a CoolSnap HQ digital camera (Photometrics). Image
analyses were performed by using ImageJ software (National Institutes of
Health). For cyclic strain experiments, computerized morphometric
analysis of fluorescence images was carried out to determine the angle
between the longest axis of the cell and the direction of applied cyclic
strain; these results are reported as the percentage of cells aligned at
90o L 30o relative to the direction of the applied strain.

[0183] For cell spreading studies, projected cell areas were measured by
tracing cell perimeters, and the areas were normalized to their
respective mean values from the earliest time point or the most compliant
substrate. For densitometric analyses of western blots, levels of GTP-Rho
were expressed as a percentage of total Rho levels, and then normalized
to baseline (control) GTP-Rho levels in normal CE cells. All data were
obtained from multiple replica experiments and are expressed as
mean±SEM. Statistical significance was determined by using Student's
unpaired t test (InStat; GraphPad).

[0184] For reorientation and scratch experiments, imaging was performed on
cells cultured on MatTek glass bottomed dishes on LEICA® Confocal
Microscope T later Cells were imaged three days after strain or stratch.

[0189] Next, the inventors transfected TEC with a human TRPV4-EGFP
construct and checked its ability to rescue aberrant mechanosensation of
tumor EC. EGFP fluorescence revealed that more than 80% cells were
transfected with TRPV4-EGFP by using the Amaxa nucleofection assay (FIG.
2A inset). TRPV4 activator, 4-α-PDD induced almost 8 fold increase
in calcium in these cells compared to EGFP alone-transfected TEC (FIG.
2A). The inventors then cultured these TRPV4-overexpressing cells on
transglutamase linked gelatin gels of various stiffness (98 to 2,280 Pa)
for 6 h and compared their cell spreading over time with that of
EGFP-transfected TEC (control). As expected and shown in FIGS. 2B and 2C,
TEC cells transfected with only EGFP attached, spread and increases their
degree of spreading with the increasing gel stiffness. They spread around
1,800 m2 on the softest (98 Pa) gel and increased their spreading
almost 2 times on gels with intermediate stiffness (370 Pa) and continued
to increase their spreading on maximal (2,280 Pa) rigidity (FIGS. 2B and
2C) confirming their abnormal mechanosensitivity. In contrast, TEC cells
transfected with TRPV4-EGFP exhibited optimal spreading on intermediate
gel stiffness and reached a plateau on maximal rigid substrate similar to
normal CE cells (Ghosh et al., 2008, PNAS, 105:11305-11310). Thus,
overexpression of TRPV4 seems to normalize the abnormal or abberant
mechanosensitivity (i.e., the requirement of stiffer substrates to
achieve maximal shape stability) of TEC.

TRPV4 Over Expression Normalizes Abnormal Angiogenesis by Tumor EC Through
the Modulation of Rho Activity and Cell Migration

[0190] Since the high basal Rho activity and dependent contractility is
the reason for abnormal mechanosensitivity of TEC (Ghosh, et al., 2008,
PNAS, 105:11305-11310), the inventors measured Rho activation and
migration of TEC. The inventors first asked if TRPV4 expression influence
cell migration on gelatin substrates. For this, the inventors have chosen
a substrate with intermediate stiffness that have been shown to support
optimal cell spreading in both TEC (control) and TEC expressing TRPV4.
Cells were cultured in the growth media were imaged and the random cell
migration was calculated. In accordance with their abnormal
mechanosensitivity, control TEC exhibited abnormal cell migration (40
μm/h) (FIGS. 3A and 3C). In contrast, TEC expressing TRPV4-EGFP
migrated slowly on these substrates (FIGS. 3 B and 3C). Similarly,
overexpression of TRPV4 also reduced migration of TEC in a scratch-wound
assay. The inventors then measured Rho activity using Rhotekin pull down
assays. The inventors found that TEC cells transfected with TRPV4-EGFP
exhibited reduced basal Rho activity (almost 50%) compared to tumor CE
cells that are expressing only EGFP (P<0.001) (FIG. 5.A and 5B)
indicating that TRPV4 expression alone inhibited or reduced basal Rho
activity in these cells. These data show that the TECs exhibited higher
baseline Rho activity and mediated contractility/migration, all of which
were reduced by expression of TRPV4.

[0191] Cell contractility and Rho activity are important mediators of
angiogenesis. The inventors have recently shown that TEC which express
high Rho activity and abnormal angiogenesis (Ghosh et al., 2008, PNAS,
105:11305-11310). Therefore, the inventors asked whether the
over-expression of TRPV4 influences TEC ability to form capillary
networks. TEC cells transfected with TRPV4-EGFP or EGFP alone were tested
for their capacity to form capillary net works using a MATRIGEL® based
in vitro angiogenesis assay. The inventors used a plating density of
8×104 cells (per well) that was shown to cause the TECs to
undergo multicellular retraction that led to gradual disruption of the
tubular network, eventually forming large cell clumps at the highest cell
plating density (REF). As expected TEC transfected with only EGFP
collapsed and failed to form tubular net work (FIG. 6A). In contrast,
overexpression of TRPV4 in TEC normalized the abnormal angiogenesis as
these cells reorganized and forms a robust multicellular capillary
network (FIG. 6B).

[0192] To confirm a direct role of TRPV4 in tumor angiogenesis in vivo,
the inventors induced tumors in TRPV4 knockout and wild type mice
(C57BL6) by subcutaneously injecting the mouse Lewis lung carcinoma cells
(LLC). the inventors found that the tumor growth was 2-3 times more in
TRPV4-/- KO mice compared to the WT mice at 21 days (FIGS. 7A and 7B).
Further, the inventors measured the tumor angiogenesis by staining the
microvessels with an endothelial specific marker, PECAM-1.
Immunohistochemical analysis revealed that the tumors from TRPV4 KO mice
exhibited significantly increased microvessel density (PECAM-1 positive)
compared to tumors from WT mice (FIG. 8). These results clearly show that
TRPV4 plays a critical role in modulating angiogenesis and absence of
TRPV4 can lead to abnormal tumor angiogenesis probably through altered
mechanotransduction by TECs.

[0193] To further confirm that TRPV4 normalizes tumor angiogenesis in
vivo, the inventors will induce tumors in wild type mice (C57BL6) by
subcutaneously injecting the mouse Lewis lung carcinoma cells (LLC).
After tumors reaching a growth of 150-200 mm3, the inventors will
give an intraperitonial injection of TRPV4 agonist (10 μg-3 mg/kg;
this is a random number) for 2-4 days followed by chemotherapeutic drugs
such as Cisplatin (3 mg/kg/week). Tumor growth will be monitored every 3
days throughout the study. The mice will be sacrificed at the end of 3
weeks and angiogenesis will be assessed by measuring microvascular
density either by immunostaining with PECAM-1 antibodies or
Alexa-conjugated isolectin. The tumors in the TRPV4 agonist treated mice
will have a reduced growth compared to placebo treated mice (control).

[0194] The references cited herein and throughout the specification are
incorporated herein by reference.